ARTICLE IN PRESS

Journal of Trace Elements in Medicine and Biology 24 (2010) 8994

Contents lists available at ScienceDirect

Journal of Trace Elements in Medicine and Biology

journal homepage: www.elsevier.de/jtemb

Nutrition

Effect of copper- and zinc-methionine supplementation on bioavailability,

mineral status and tissue concentrations of copper and zinc in ewes
D.T. Pal n, N.K.S. Gowda, C.S. Prasad 1, R. Amarnath, U. Bharadwaj, G. Suresh Babu, K.T. Sampath
National Institute of Animal Nutrition and Physiology, Adugodi, Bangalore 560 030, India

a r t i c l e in fo

abstract

Article history:
Received 9 January 2008
Accepted 30 November 2009

The effect of feeding Cu- and Zn-methionine to ewes was studied in a 240d feeding trial. The plasma
and tissue Cu and Zn concentrations and Cu/Zn-superoxide dismutase (Cu/Zn-SOD) activity were
employed to assess the relative bioavailability from Cu- and Zn-methionine. The macro and
micronutrient intake, utilization, plasma mineral status, tissue accumulation of Cu and Zn as well as
wool concentration of Cu and Zn were studied in ewes (n =12) fed a corn-soybean meal based basal
diet with 50% more Cu and Zn supplementation over the basal diet either from Cu- and Zn-sulfate
(Cu-Sulf+ Zn-Sulf group) or Cu- and Zn-methionine (Cu-Meth +Zn-Meth group). The average daily feed
intake and body weight gain of ewes did not differ due to dietary supplementation of Cu- and
Zn-methionine. However, dry matter intake was comparatively lower and thus resulted in better feed:
gain in Cu- and Zn-methionine group as compared to ewes fed Cu- and Zn-sulfate. Supplementation of
Cu and Zn over the basal diet either from methionine-chelated or sulfate sources resulted in increased
plasma Cu and Zn as well as Cu/Zn-SOD activity on d-30, which indicated a positive correlation between
plasma Cu and Zn and Cu/Zn-SOD activity. The gut absorption, liver concentrations of Cu and Zn, and
liver Cu/Zn-SOD activity were signicantly (P o0.01) higher in ewes supplemented with Cu- and
Zn-methionine compared to Cu- and Zn-sulfate. Periodical analysis of wool samples indicated no
signicant difference in Cu and Zn content between Cu-and Zn-methionine and Cu- and Zn-sulfate
groups. Feeding of Cu and Zn from methionine-chelated source resulted in reduced (P o 0.01) excretion
of Cu and Zn in feces indicating their better utilization, and this will have positive implication on
environment. The gut absorption values, plasma and liver tissue concentrations of Cu and Zn supported
the hypothesis that Cu- and Zn-methionine supplements have better bioavailability compared to
Cu- and Zn-sulfate and Cu- and Zn-dependent enzyme (Cu/Zn-SOD) could be used to determine the
bioavailability of Cu and Zn.
& 2009 Elsevier GmbH. All rights reserved.

Keywords:
Bioavailability
Copper-methionine
Superoxide dismutase
Tissue minerals
Zinc-methionine

Introduction
Copper (Cu) and zinc (Zn) are critical trace minerals for
production and reproduction in farm animals. The inorganic form
of trace minerals are not sufciently absorbed and retained.
However, the organic form of these minerals are absorbed
efciently and retained in the tissue to enhance performance,
improve immunity, health and reproduction compared to inorganic forms [1]. Limited research has been done concerning the
biological availability of organic mineral sources in sheep. In
studies with cattle [24], pigs [5], rats [6] and ewes [7,8] tissue
concentrations of Cu and Zn were found higher for organic than
inorganic sources. Lambs supplemented with Zn in the diet from
Zn-lysine had higher Zn concentration in different tissues than
lambs received Zn-sulfate or Zn-oxide [7,9,10].

Availability of trace minerals from common feeds is variable

from poor to levels that can interfere with absorption or
utilization of other minerals. Cu and Zn are the most limiting
trace minerals under farm conditions [11]. The aim of the present
study was to evaluate the effect of supplementing Cu- and
Zn-methionine sources on gut absorption, plasma Cu and Zn
status, tissue concentrations of Cu and Zn, and relative bioavailability as compared to inorganic source of Cu and Zn (Cu- and
Zn-sulfate) in ewes and to ascertain the utility of Cu/Zn-superoxide dismutase (Cu/Zn SOD), as a biochemical marker to determine the bioavailability of Cu and Zn from different sources.

Materials and methods

Animals and experimental design

Corresponding author. Tel.: + 91 80 25711304; fax: + 91 80 25711420.

E-mail address: dtpal@yahoo.co.in (D.T. Pal).
1
ICAR, Krishi Bhavan, New Delhi 110 001, India.

0946-672X/$ - see front matter & 2009 Elsevier GmbH. All rights reserved.
doi:10.1016/j.jtemb.2009.11.007

Animal experimental protocol was approved by the Institute

Ethical Committee, NIANP, Bangalore, India. Twelve ewes based

ARTICLE IN PRESS
90

D.T. Pal et al. / Journal of Trace Elements in Medicine and Biology 24 (2010) 8994

Table 1
Dietary composition of basal diet (%).
Ingredients

Concentrate feed

Para grass

Crushed corn
Soybean meal
Calcium carbonate
Sodium chloride
Mineral mixturea
Vitamin A, D and Eb

55.00
43.50
1.00
0.50
0.2
0.02

Chemical composition (% DM basis)

OM
CP
Ash
AIA
Ca
P
Mg
Cu (ppm)
Zn (ppm)

94.55
16.82
5.45
0.59
0.56
0.69
0.34
14.66
94.17

89.26
6.05
10.74
4.20
0.57
0.34
0.40
8.67
76.40

a
Mineral mixture consists of 25 mg of Fe as FeSO4, 20 mg of Mn as MnSO4 and
0.1 mg of Co as CoCl2 without Cu and Zn salts per kg of diet.
b
Provided 5000 IU of vitamin A, 500 IU of vitamin D3 and 187 mg of vitamin E
per kg of diet.

on their body weight randomly assigned to two groups of six

animals each. One group was supplemented with 50% more of Cu
and Zn over the basal diet from feed grade inorganic source
(copper sulfate and zinc sulfate; Cu-Sulf+ Zn-Sulf group) and the
other group was supplemented with 50% more of Cu and Zn
over the basal diet from commercially available feed grade
organic source (copper-methionine and zinc-methionine;
Cu-Meth+Zn-Meth group). The ewes were fed a corn-soybean
meal and green para grass based basal diet (9.8 mg of Cu and
39.33 mg of Zn per kg of DM). The diet was formulated to be
adequate in protein, energy, vitamins and minerals for this
category of sheep [12]. Ingredient composition of concentrate
feed is provided in Table 1. All ewes were maintained under
uniform managerial conditions by housing them in a wellventilated shed with facilities for individual feeding. The ewes
were dewormed with anthelmintic and were vaccinated for
common diseases before the experiment. Ewes were individually fed the dry matter (DM) at 3% of their body weight to
meet the nutrient requirement as per ICAR [12]. Fresh drinking
water was made available at all the time. Water samples were
collected at the beginning, middle and end of the study,
composited and analyzed for Cu and Zn. Body weight of ewes
was recorded at monthly interval till end of the study. A
digestibility trial of six days duration was conducted to study
the nutrient utilization and gut absorption of minerals in ewes fed
organic and inorganic sources of Cu and Zn in the diet.

Sample collection
The concentrate feed was provided to ewes at 08.30 h daily,
chaffed green para grass (Brachiaria mutica) was offered in the
afternoon. Residue if any was weighed next morning to calculate
the feed intake by the ewes.
Representative samples of green para grass and concentrate
feed were collected daily and dried in hot air oven at 105 1C to
determine the DM content and calculate the daily DM intake by
ewes.
Blood samples were collected on d 0 of the experiment and
then subsequently at d 30, 60, 90, 120, 150, 180, 210 and 240 of
the experimental period via jugular venipuncture in heparinizedtrace mineral free tubes. The whole blood was centrifuged at 800g
for 10 min, plasma was aspirated into multiple polyethylene

tubes and frozen at

20 1C until analyzed for minerals and
enzyme. Wool samples were collected by clipping 4  4 cm2 area
from rib region at d 0, 30, 120 and 240. The dirt and grease of wool
samples were removed by thoroughly rinsing the wool rst with
detergent followed by ethanol. The wool was nally washed
several times in bidistilled water, oven dried at 60 1C for 24 h and
kept in sealed plastic bags for minerals analysis.
At the middle of the experiment (d 160), a digestibility trial
was conducted to determine the nutrient utilization in ewes. The
feed and fecal samples of the trial were collected and processed
daily for DM estimation in hot air oven and then ground in a willy
grinding machine to pass through 1 mm mesh and stored in
plastic containers for further analysis.
All the ewes were slaughtered at the end of the study and
tissue samples of liver, kidney, heart, lung and muscle (Biceps
femoris) from femoral region and skin from lower front leg were
collected and frozen at 20 1C for further mineral and enzyme
analysis. Wool was shaved from each skin sample, and visible
connective tissue was separated from the skin and preserved at
20 1C for further analysis.
Chemical analysis
Total ash and crude protein estimation
Total ash and acid insoluble ash of the experimental diets and
fecal samples were estimated as per the method of A. O. A. C. [13],
whereas the protein content in feed and feces was estimated in
semi-automatic Kjel Plus systems (Pelican Equipments, Chennai,
India). Reference material used was puried casein (Titan Biotech
Indian Pvt. Ltd., New Delhi, India) for standardization of protein
content in samples and crude protein content in standard casein
was 87.30% on dry weight basis.
Mineral analysis
Samples were analyzed for minerals in atomic absorption
spectrophotometer (Perkin Elmer AA Analyst 300, USA). Samples
of feeds, fodders and feces were wet digested with 36 N nitric acid
and 70% perchloric acid (3:1) in a microwave digestion system
(Multiwave 3000, Anton Paar, USA) operated using a software at
15 bar pressure and 190 1C temperature with 10 min ramp and
30 min holding time.
Wool and tissue samples (0.2 g) were also wet digested with
nitric acid and perchloric acid (3:1) at 15 bar pressure and 190 1C
temperature with 8 min ramp and 19 min holding times in the
microwave digestion system. After completion of digestion, the
clear extract obtained was transferred to volumetric ask and
volume made up to 100 mL with deionized water for analysis of
macro and micro minerals.
Calcium (Ca), magnesium (Mg), copper (Cu) and zinc (Zn) in
samples were estimated in atomic absorption spectrophotometer
using air-acetylene ame. For estimation of Ca and Mg in samples,
acid extracts were suitably diluted with 0.1% lanthanum chloride
to avoid interference from phosphates and other minerals. Copper
and zinc in feed and fecal samples were analyzed either directly
from acid extract without any further dilution or diluted with
deionized water, if required. Phosphorus in feed, feces and plasma
samples was analyzed by colorimetric method [14] using
stannous chloride as a reducing agent. Mineral standards
(National Institute of Standards and Technology certied) were
run in each analysis to ensure the accuracy of estimation.
Estimation of Cu/Zn-superoxide dismutase enzyme activity
Cu-and Zn-dependent enzyme, (Cu/Zn-superoxide dismutase,
Cu/Zn-SOD) activity was determined in liver tissue and blood

ARTICLE IN PRESS
D.T. Pal et al. / Journal of Trace Elements in Medicine and Biology 24 (2010) 8994

plasma. Liver homogenate preparation was performed on the day

of enzyme analysis. Homogenate was centrifuged at 800g for
10 min, supernatant removed and the pellet resuspended in
0.25 mol/L sucrose buffer to a nal concentration of 250 g/L.
Homogenate was diluted to a nal concentration of 0.5 g/L for
analysis of Cu/Zn-SOD activity.
The Cu/Zn-SOD activity in liver tissue and plasma was
determined by a modied method of pyrogallic acid autoxidation
[15]. The change in absorbance/minute in control (without
sample) and test was recorded to calculate the SOD activity.
One unit of SOD activity was expressed as amount of enzyme
required to inhibit the pyrogallol autoxidation by 50%, and
activity was expressed as units of enzyme per mg of fresh tissue.
Bioavailability estimation of Cu and Zn
Bioavailability of Cu and Zn from organic sources was
determined from gut absorption, Cu/Zn-SOD activity as well as
from Cu and Zn content of liver tissue. Regression procedure was
used to calculate the bioavailability of Cu- and Zn-methionine
relative to that for feed grade Cu-and Zn-sulfate as per the method
described by Cao et al. [9].

91

the means was compared using standard Fisher (F)-values and

compared statistically at 1% and 5% level of signicance. Relative
bioavailability values were determined by slope ratio comparison
from multiple linear regression [17] using copper sulfate and zinc
sulfate as standard sources.

Results
Feed intake and weight gain
Dietary ingredient and nutrient composition of basal diet is
presented in Table 1. The para grass contained 8.67 mg Cu and
76.4 mg Zn per kg DM. The Cu and Zn content of drinking water
was negligible and hence was not accounted for calculating the
intake. The concentrate mixture used in this study contained
16.82% crude protein and 14.66 mg Cu and 94.17 mg Zn per kg
DM. The average daily feed intake and daily weight gain did not
differ amongst the groups. However, ewes supplemented with
organic source of Cu and Zn consumed 7.5% less feed over 3060 d
period at targeted body weight gain, resulting in better (P40.05)
feed conversion efciency (Table 2).

Statistical analysis
The study was conducted as a completely randomized design
with ewe as the experimental unit. The effect between the
subjects was dietary sources of Cu and Zn. For plasma minerals
and Cu/Zn-SOD activity, the study was conducted as a completely
randomized design in a factorial arrangement of treatments with
repeated measurements, where effect between the subjects was
dietary sources of Cu and Zn and effect within subjects was
sampling time. The interaction was dietary source and time. The
data of various parameters were subjected to one-way ANOVA
using the dietary source as a variable [16]. The difference between

Plasma mineral prole

Plasma Cu and Zn concentration was signicantly higher
(P o0.06) in ewes that consumed Cu-Meth+ Zn-Meth compared to
those supplemented with Cu-Sulf+ Zn-Sulf sources. The source
and period interaction for plasma Cu and Zn was signicant and
was signicantly higher (Po0.05) in ewes fed Cu-Meth+ Zn-Meth,
than those supplemented Cu-Sulf+ Zn-Sulf during 30 and 60d of
supplementation (Fig. 1). Calcium, phosphorus and magnesium
concentrations in blood plasma were statistically (P40.05)
similar between inorganic (Cu-Sulf+ Zn-Sulf) and organic
(Cu-Meth+ Zn-Meth) Cu and Zn supplemented group throughout
the experimental period.

Table 2
Body weight, average daily gain and feed: gain of ewes.

Nutrient utilization and gut absorption

Attributes

Cu-Sulf +
Zn-Sulf

Cu-Meth+
Zn-Meth

SD

P-value

Initial body weight (kg)

Final body weight (kg)
Body weight gain (kg)
Average daily gain (g/d)
Feed intake (g/d)
Feed: gain

15.17
22.85
7.68
36.60
412.70
11.8:1

15.00
22.77
7.77
37.00
381.81
10.3:1

2.97
3.46
1.31
6.25
60.35
2.61

0.928
0.969
0.919
0.918
0.401
0.349

4.00

Cu-Sulf & Zn-Sulf

Cu-Meth & Zn-Meth

2.50

Cu-Sulf + Zn-Sulf
Cu-Meth + Zn-Meth

3.50
Plasma Zn (mg/L)

Plasma Cu (mg/L)

3.00

No signicant difference was observed in intake and utilization

of macronutrients and macro minerals on supplementing either
inorganic or organic source of Cu and Zn. However, the gut
absorption of Cu and Zn was signicantly (Po0.01) higher in
ewes fed diets with organic Cu and Zn compared to inorganic
source, resulting in reduced fecal excretion of these minerals
(Table 3).

2.00
1.50
1.00
0.50

3.00
2.50
2.00
1.50
1.00
0.50

0.00

0.00
0

Period of collection (Month)

1 2 3 4 5 6 7
Period of collection (Month)

Fig. 1. Effect of dietary supplementation of Cu- and Zn-methionine on plasma Cu and Zn level in ewes.

ARTICLE IN PRESS
92

D.T. Pal et al. / Journal of Trace Elements in Medicine and Biology 24 (2010) 8994

Table 3
Effect of dietary supplementation of Cu- and Zn-methionine on intake and gut absorption of Cu and Zn in ewes.
Attributes

Cu-Sulf +Zn-Sulf

Cu-Meth+Zn-Meth

P-value, signicance

Copper
Total intake (mg/d)
Excreted (mg/d)
Gut absorption (mg/d)
Gut absorption (%)

8.48
6.63
1.85
21.88

8.10
5.42
2.65
33.11

0.69
0.97
0.62
8.19

0.323, NS
0.022n
0.019n
0.009nn

Zinc
Total intake (mg/d)
Excreted (mg/d)
Gut absorption (mg/d)
Gut absorption (%)

45.30
31.67
13.62
30.12

42.33
25.24
17.10
40.40

5.07
5.02
3.07
6.71

0.334, NS
0.010nn
0.034n
0.000nn

81.05
381.27

68.77
304.20

9.47
53.53

Fecal Cu and Zn
Fecal Cu (ppm)
Fecal Zn (ppm)
NS, non-signicant; nP o0.05;

Tissue

Cu-Sulf + Zn-Sulf

0.016nn
0.005nn

P o0.01.

nn

Table 4
Effect of Cu- and Zn-methionine supplementation on tissue Cu and Zn concentrations (mg/g) in ewes.

SD

Cu-Meth+Zn-Meth

SD

P-value,
signicance

Copper
Livern
Kidney
Heart
Muscle
Skin

230.00
17.66
23.00
6.73
10.60

325.00
17.33
23.50
6.07
10.43

91.365
0.797
1.864
1.097
3.594

0.048n
0.496, NS
0.664, NS
0.266, NS
0.940, NS

Zinc
Livern
Kidney
Heart
Muscle
Skin

111.67
108.33
92.67
120.67
49.83

128.33
106.50
97.17
120.17
49.00

13.554
10.706
12.325
27.401
10.094

0.023n
0.782, NS
0.553, NS
0.977, NS
0.894, NS

Table 5
Cu and Zn concentrations (mg/g) of wool in ewes fed on Cu- and Zn-methionine
sources.
Attribute

Cu

Zn

Effect of source
Cu-Sulf + Zn-Sulf
Cu-Meth+ Zn-Meth
Signicance
P-value

12.20
11.72
NS
0.377

115.73
117.40
NS
0.691

10.97a
11.22ab
11.70abc
12.83bc
13.08c

108.17a
109.67a
110.00a
123.33b
131.67b

nn

Effect of periodn
0d
60 d
120 d
180 d
240 d
Signicance
P-value
SEM

0.037
0.271

0.000
2.070

Po 0.05
Signicance level nP o0.05,

Copper and zinc content in liver and wool

The Cu and Zn content in liver was signicantly (P o0.05)
higher in ewes fed diets containing organic sources (Cu and
Zn-methionine). No signicant differences were observed in Cu
and Zn concentrations in other tissues like heart, kidney, muscle
and skin (Table 4). The content of Cu and Zn in wool was not
inuenced by the dietary supplementation of Cu-methionine and
Zn-methionine, but when compared with the values of Cu and Zn
on day 0, accumulation of these trace minerals was quicker and
signicantly (P o0.05) higher in ewes supplemented with organic
forms than the inorganic forms (Table 5).

Activity of Cu/Zn-superoxide dismutase and relative bioavailability

The activity of Cu/Zn-SOD in liver homogenate was signicantly (Po0.01) higher in ewes fed Cu and Zn-methionine and
was positively correlated with Cu and Zn content in liver (Fig. 2).
The bioavailability values for Cu and Zn were higher in ewes fed
Cu and Zn-methionine compared to CuSO4 and ZnSO4 and found
to be 151% and 134%, respectively. Relative bioavailability
estimate based on multiple regression slope ratios of Cu/Zn-SOD
regressed on plasma level of Cu and Zn was found to be 150% and
132%, respectively. Bioavailability of Cu and Zn determined from
the liver Cu and Zn content and liver SOD activity was estimated
at 151% and 133%, for Cu and Zn, respectively. It was found that

nn

Po 0.01.

the bioavailability determined from gut absorption, enzyme

activity and tissue deposition of Cu and Zn were similar
(Table 6, Figs. 2 and 3).

Discussion
The ewes supplemented with organic source of Cu and Zn
showed better feed conversion efciency. Similar observations
have been reported by other workers [18,8,19], suggesting better
nutrient utilization. Higher plasma levels of Cu and Zn in ewes fed
Cu-and Zn-methionine was attributed to better gut absorption of
these minerals. Absorption and transportation of Zn-methionine
in intact form following oral dosing and slower rate of decline in
plasma level has been reported by Spears [20].
Higher gut absorption and lower fecal excretion of Cu and Zn
from organic source with similar intake in the present study is in
agreement with the nding of Power and Horgan [23] and it could
be believed that the mineral ions from inorganic source in gut
are released and possibly recombine with other digesta components forming insoluble complexes, thus reducing their gut
absorption, while the organic minerals use amino acid uptake
mechanism across the mucosa and absorbed to blood completely.
Mondal et al. [24] also reported lower excretion of Cu through
Cu-proteinate as compared to CuSO4.
The higher Cu and Zn levels in the present study for initial
period (6090 d) followed by comparable levels to that of ewes

ARTICLE IN PRESS

250000

y = 267.98x + 81382

200000
150000
100000
50000
100 150 200 250 300 350 400
Liver Cu (mg/kg)
Cu-Sulf + Zn-Sulf

300000
250000

y = 1057.5x + 34985

200000
150000
100000
50000
90

100

110

120

130

Liver SOD activity (Unit)

Cu-Sulf + Zn-Sulf
300000

93

Cu-Meth + Zn-Meth

300000

y = 406.79x + 81240

250000
200000
150000
100000

50000
100 150 200 250 300 350 400 450
Liver Cu (mg/kg)

Liver SOD activity (Unit)

Liver SOD activity (Unit)

Liver SOD activity (Unit)

D.T. Pal et al. / Journal of Trace Elements in Medicine and Biology 24 (2010) 8994

Cu-Meth + Zn-Meth

300000
250000

y = 1408x + 32748

200000
150000
100000
50000
90 100 110 120 130 140 150 160

Liver Zn (mg/kg)

Liver Zn (mg/kg)

Fig. 2. Relationship between liver Cu and Zn and liver SOD activity in ewes fed Cu and Zn from sulfated and methionine-chelated sources.

Table 6
Relative bioavailability values (RBV) of Cu and Zn estimated based on multiple
regression slope ratios and from gut absorption.
Attribute

Liver
Cu
Zn

RBV (%)n

Slope
Cu-Sulf + Zn-Sulf

Cu-Meth+Zn-Meth

267.98
1057.5

406.79
1408.0

151.79
133.14

Plasma
Cu
Zn

11.675
10.447

17.522
13.836

150.08
132.44

Gut absorption (%)

Cu
Zn

21.88
30.12

33.11
40.40

151.32
134.13

Assumed that bioavailability of Cu and Zn from Cu- and Zn-sulfate was 100%.

fed organic source indicated the deposition of these minerals in

soft tissues as a part of the homeostatic mechanism. This is
evident from the higher Cu and Zn content in liver of ewes fed
organic source. Further signicantly higher activity of Cu/Zn-SOD
in liver tissue substantiates the better bioavailability of Cu/Zn
from organic source and also explains the Cu and Zn dependent
activity of SOD. Andrewartha and Caple [21] also reported similar
relationship of SOD with serum Cu. Suttle and Mc Murray [22]
recorded a temporal relationship of Cu level with SOD of RBC and
hence could be of diagnostic signicance, while evaluating the Cu
status. In studies with cattle [2,3], pig [5], rat [6] and ewes [8], the
liver Cu and Zn contents were higher in organic Cu and Zn group.
Wright and Spears [10] also reported higher Zn concentration in
liver and kidney of calves fed Zn-proteinate and Zn-methionine
compared to ZnSO4. These ndings support the higher gut
absorption and bioavailability of trace minerals from organic
sources.
When compared to Cu and Zn values of wool at day 0,
accumulation of these minerals was signicantly (Po0.05) higher
in ewes supplemented with Cu and Zn-methionine. Similar
ndings have been reported by Ryan et al. [25]. Eckert et al.
[26] reported the improvement in wool quality in terms of wool
length due to feeding of Cu-proteinate.

Bioavailability indicates the absorption and utilization of a

nutrient. Mile and Henry [27] used absorption of a mineral
as an indicator of its bioavailability. In some cases certain
metabolically essential compounds or enzymes have been used
as criteria for assessing mineral bioavailability [28,29]. The
results of relative bioavailability of Cu and Zn from gut absorption,
plasma Cu and Zn levels, Cu and Zn content in liver and
Cu/Zn-SOD activity, indicated similarities, suggesting that these
parameters can be used for determining the bioavailability of Cu
and Zn.
Cao et al. [9] reported the Zn bioavailability values of 130%,
110% and 113% for Zn-proteinate, Zn-amino acid chelate and
Zn-methionine, respectively, as compare to zinc sulfate in lambs.
In the present study, the ewes were fed a diet where corn (which
contains high phytate) was one of the major feed ingredients and
para grass as bre source. Hence, there might be the possibility
for interaction of minerals especially, Zn with phytate when
supplemented through inorganic sources in the digestive tract
and thus reduced the bioavailability of Cu and Zn. Higher phytate
and bre contents in the diet known to reduce the bioavailability
of trace minerals by interacting negatively [30] whereas, the
above minerals when supplemented through organic sources such
negative effects of mineral interaction did not happen as minerals
were chelated with amino acid and remained inert in any
chemical reactions and thus improved the bioavailability of Cu
and Zn.

Conclusions
Supplementation of Cu and Zn through organic sources
(Cu-methionine, Zn-methionine) as compared to inorganic
sources (CuSO4, ZnSO4) improved gut absorption, higher activity
of Cu/Zn-SOD and tissue retention of Cu and Zn, suggested better
bioavailability through organic/chelated sources. Further, the
results have shown that mineral-dependent enzymes like
Cu/Zn-SOD can be used as a biomarker for assessing the status
of Cu and Zn. Higher bioavailability of trace minerals through
organic sources effectively reduces their dietary requirement and
hence, is an environment friendly approach in reducing the fecal
excretion of minerals.

ARTICLE IN PRESS
94

D.T. Pal et al. / Journal of Trace Elements in Medicine and Biology 24 (2010) 8994

Cu-Sulf + Zn-Sulf

Cu-Meth + Zn-Meth

y = 11.675x + 7.9193

25

SOD activity (Unit)

SOD activity (unit)

30
20
15
10
5
0
0

0.5

1.5

40
35
30
25
20
15
10
5
0

y = 17.522x + 0.2389

0.5

Plasma Cu (mg/L)
Cu-Sulf + Zn-Sulf

25
20
15
10
5
0
0

0.5
1
1.5
Plasma Zn (mg/L)

2.5

Cu-Meth + Zn-Meth

y = 10.447x + 8.9212

SOD activity (Unit)

SOD activity (Unit)

30

1
1.5
2
Plasma Cu (mg/L)

45
40
35
30
25
20
15
10
5
0

y = 13.836x + 4.8113

0.5

1
1.5
2
Plasma Zn (mg/L)

2.5

Fig. 3. Plasma Cu and Zn level and Cu/Zn-SOD activity in ewes fed on Cu- and Zn-methionine.

Acknowledgements
This work has been carried out under the All India Coordinated
Research Project (AICRP) of Indian Council of Agricultural Research
(ICAR), New Delhi. The facilities provided by Director, National
Institute of Animal Nutrition and Physiology (NIANP), Bangalore
for conducting this research is thankfully acknowledged.
References
[1] Spears JW. Organic trace minerals in ruminant nutrition. Anim Feed Sci
Technol 1996;58:15163.
[2] Nockels CF, Debonis J, Torrent J. Stress induction affects copper and zinc
balance in calves fed organic and inorganic copper and zinc sources. J Anim
Sci 1993;71:253945.
[3] Rojas LX, McDowell LR, Martin FG, Wilkinson NS, Johnson AB, Njeru CA.
Relative bioavailability of zinc methionine and two inorganic zinc sources fed
to cattle. J Trace Elem Med Biol 1996;10(205-209):34.
[4] Spears JW. Trace mineral bioavailability in ruminants. J Nutr 2003;133:
1506S1509S.
[5] Apgar GA, Kornegay ET, Lindrmann MD, Notter DR. Evaluation of copper
sulfate and a copper lysine complex as growth promoters for weanling swine.
J Anim Sci 1995;73:26406.
[6] Du ZR, Hemken W, Jackson JA, Trammel DS. Utilization of copper in copper
proteinate, copper lysine and cupric sulfate using the rat as experimental
model. J Anim Sci 1996;74:165763.
[7] Rojas LX, McDowell LR, Cousins RJ, Martin FG, Wilkerson NS, Johnson ABq,
et al. Relative bioavailability of two organic and two inorganic sources fed to
sheep. J Anim Sci 1995;73:1202.
[8] Hateld PG, Swenson CK, Kott RW, Ansotegui RP, Roth NJ, Robinson BL. Zinc
and copper status in ewes supplemented with sulfate- and amino acid
complexed forms of zinc and copper. J Anim Sci 2001;79(1):2616.
[9] Cao J, Henry PR, Guo R, Holwerda RA, Toth JP, Littell RC, et al. Chemical
characteristics and relative bioavailability of supplemental organic zinc
sources for poultry and ruminants. J Anim Sci 2000;78:203954.
[10] Wright CL, Spears JW. Effects of zinc source and dietary level on zinc
metabolism in Holstein bull calves. J Anim Sci 2001;79(suppl. 1):8692.
[11] Gowda NKS, Ramana JV, Prasad CS, Singh K. Micronutrient content of certain
tropical conventional and unconventional feed resources of Southern India.
Trop Anim Health Prod 2004;36:7794.
[12] ICAR. Indian Council of Agricultural Research. Nutrient Requirements of Livestock
and Poultry 1998. 2nd ed., ICAR, DIPA, Krishi Bhavan, New Delhi, India.
[13] AOAC. Ofcials methods of analysis (14th ed.). Association of Ofcial
Analytical Chemists. Washington D C.1990.
[14] Fiske CH, Subbarow Y. The colorimetric determination of phosphorus. J Biol
Chem 1925;66:375400.

[15] Percival SS. Cu/Zn superoxide dismutase activity does not parallel copper levels
in copper supplemented LH-60 cells. Biol Trace Elem Res 1993;38:6372.
[16] Snedecor G.W., Cochran W.G. Statistical Methods. 8th Ed. (Oxford and
IBH Publishing Co., Calcutta) Iowa State University Press Ames, Iowa, USA.
1994.
[17] Littell RC, Lewis AJ, Henry PR. Statistical evaluation of bioavailability assays.
In: Ammerman CB, Baker DH, Lewis AJ, editors. Bioavailability of Nutrients
for Animals: Amino Acids, Minerals, and Vitamins. San Diego, CA: Academic
Press; 1995. p. 533.
[18] Ahola JK, Baker DS, Burns PD, Mortimer RG, Enns RM, Whittier JC, et al. Effect
of copper, zinc and manganese supplementation and source on reproduction,
mineral status and performance in growing beef cattle over a two-year
period. J Anim Sci 2004;82:237583.
[19] Wright CL, Spears JW. Effect of zinc source and dietary level on zinc
metabolism in Holstein calves. J Dairy Sci 2004;87:108591.
[20] Spears JW. Zinc methionine for ruminants: relative bioavailability of zinc in
lambs and effects of growth and performance of growing heifers. J Anim Sci
1989;67:83543.
[21] Andrewartha KA, Caple IW. Effects of changes in nutritional copper
on erythrocyte cuperoxidase dismutase activity in sheep. Res Vet Sci
1980;28(1):1014.
[22] Suttle NF, McMurray CH. Use of erythrocyte copper zinc superoxide
dismutase activity and hair or eece copper concentrations in the diagnosis
of hypocuprosis in ruminants. Res Vet Sci 1983;35:4752.
[23] Power R, Horgan K. Biological chemistry and absorption of inorganic
and organic trace metals. In: Lyons TP, Jacques KA, editors. Biotechnology in the Feed Industry. Nottingham, UK: Nottingham University Press;
2000. p. 27791.
[24] Mondal MK, Biswas P, Roy B, Mazumdar D. Effect of copper sources and levels
on serum lipid proles in Black Bengal (Capra hircus) kids. Small Rum Res
2007;67:2835.
[25] Ryan JP, Kearns P, Quinn T. Bioavailability of dietary copper and zinc in adult
Texel sheep: a comparative study of the effect of sulphate and Bioplex
supplementation. Ir Vet J 2002;5(5):2214.
[26] Eckert GE, Greene LW, Carstens GE, Ramsey WS. Copper status of ewes fed
increasing amounts of copper from copper sulfate or copper proteinate. J
Anim Sci 1999;77:2449.
[27] Miles RD, Henry PR. Relative trace mineral bioavailability. Cienc Anim Bras
2000;1(2):7393.
[28] Ammerman CB, Henry PR, Miles RD. Supplemental organically-bound
mineral compounds in livestock nutrition. In: Garnsworthy PC, Wiseman. J,
editors. Recent Advances in Animal Nutrition. Nottingham, UK: Nottingham
University Press; 1998. p. 6791.
[29] Ammerman C.B., Henry P.R., Miles R.D. Bioavailability of organic forms of the
microminerals. Proceedings of the 9th Annual Florida Ruminant Nutrition
Symposium. Gainesville, Florida 1998b; p. 33-49.
[30] Wedekind KJ, Lewis AJ, Giesmann MA, Miller PS. Methodology of assessing
zinc bioavailability: efcacy estimates for zinc-methionine, zinc sulfate, and
zinc oxide. J Anim Sci 1994;70:17887.