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Effects of Copper and Zinc Supplementation on Weight Gain
and Hematological Parameters in Pre-weaning Calves
Guillermo Alberto Mattioli
1
&Diana Esther Rosa
1
&Esteban Turic
2
&Alejandro Enrique Relling
1
&Esteban Galarza
1
&
Luis Emilio Fazzio
1
Received: 5 December 2017 /Accepted: 29 December 2017
#Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
Cow-calf operations may be affected by trace mineral deficiencies, particularly copper (Cu) and zinc (Zn) deficiency, which may
decrease the calf daily weight gain and alter hematological parameters. We evaluated the effect of Cu and Zn supplementation on
pre-weaning calves (n= 40; 92 ± 6 kg initial body weight) from the Salado River basin, Buenos Aires, Argentina. Calves were
divided into four groups (n= 10 each) and subcutaneously administered 0.3 mg/kg Cu (Cu group), 1 mg/kg Zn (Zn group), Cu
and Zn together (Cu + Zn group), and sterile saline solution (control group) every 40 days for 120 days. Plasma Cu and Zn
concentrations, hematological parameters, and weight were recorded every 40 days. A completely randomized 2 × 2 factorial
treatment design was used and data were analyzed with a mixed model for repeated measures over time. Cu and Zn were detected
in plasma after the second sampling. Cu × Zn interaction was significant (p= 0.09), being Cu concentration higher in the Cu + Zn
than in the Cu group. Differences in weight gain (Zn × time interaction; p< 0.01) were observed in the Zn but not in the Cu group
(p> 0.1). On the other hand, none of the treatments altered any of the hematological parameters assessed (p>0.1).Our results
show the risk of lower weight gain due to Zn deficiency in pre-weaning calves raised in the Salado River basin.
Keywords Copper .Zinc .Deficiency .Weight gain .Hematological parameters
Introduction
Trace minerals provide the essential nutrients animals need for
physiological functions, such as growth and development, im-
munity, and reproduction. Consequently, their deficiency can
negatively affect animal performance [1,2]. Copper (Cu) defi-
ciency is the second most frequent mineral deficiency in grazing
cattle worldwide, causing considerable production losses in well-
characterized areas [3]. On the other hand, zinc (Zn) deficiency is
involved in health problems associated with the immune system
and reproductive losses, as well as growth and integrity of the
skin and hooves; however, the pathogenesis remains poorly un-
derstood [3].
In the province of Buenos Aires, Argentina, beef cattle
production represents the main economic activity of the
Salado River basin (SRB). This area covers 5.5 million hect-
ares and produces two million calves a year [4]. Animals are
raised under an extensive system based on naturalized grass as
the main source of nutrients. The economic benefit of the
region resides on selling calves weaned at 6–7monthsofage.
Different authors have reported Cu and Zn deficiency in the
SRB [5–7], together with related effects such as decreased
dailyweightgain[3,8,9] and hematological changes [10].
Although the diagnosis of both deficiencies in the herd is
based on the assessment of plasma Cu and Zn concentrations,
there are discrepancies regarding data interpretation. In terms
of Cu, concentrations > 57 μg/dL are considered adequate and
<57 μg/dL indicate a deficiency [3]. Nevertheless, these au-
thors reported clinical symptoms of hypocuprosis with Cu
levels < 30 μg/dL [3]. In contrast, Cu concentrations of 50–
70, 20–50, and < 20 μg/dL are considered as marginal, defi-
cient, and clinically evident disease, respectively [11].
Regarding Zn, concentrations > 90 μg/dL are considered to
be adequate, 80–90 μg/dL as marginal, and < 80 μg/dL as
deficient [2]. Both Cu and Zn deficiencies are associated with
*Guillermo Alberto Mattioli
mattioli@fcv.unlp.edu.ar
1
Laboratorio de Nutrición Mineral, Facultad de Ciencias Veterinarias,
Universidad Nacional de La Plata, 60 y 118, 1900 La
Plata, Argentina
2
Biogenésis Bagó, Buenos Aires, Argentina
Biological Trace Element Research
https://doi.org/10.1007/s12011-017-1239-0
hematological changes such as anemia, leukopenia, and al-
tered tissue enzymes [10,12].
Here we discuss whether plasma Cu and Zn concentrations
are increased after parenteral Cu, Zn, and Cu + Zn supplemen-
tation of pre-weaning calves, thereby altering daily weight
gain and hematological parameters.
Materials and Methods
All experimental procedures were approved by the Committee
for the Care and Use of Laboratory Animals (CICUAL, for its
Spanish acronym), School of Veterinary Sciences, La Plata
National University, Argentina (Protocol no. 58-2-16P).
The trial was carried out on the experimental farm
BManantiales,^located in Chascomús, Buenos Aires (35°
44′31.5^S58°06′11.7^W). The characteristics of the farm
are comparable to those in the SRB, including poor drainage,
floods, and higher quantity and quality grass production dur-
ing spring.
Animals
A total of 40 clinically healthy Aberdeen Angus calves were
used. They were kept as cow-calf pairs since time 0 of the trial
(3 months of age) until weaning (7 months of age; time 120 of
the trial). The vaccination program of calves included foot-
and-mouth disease vaccine and two doses of clostridial vac-
cine before weaning. Gastrointestinal parasites were monthly
examined through fecal egg counts.
The animals were fed on native and naturalized grass
(Chaetotropis elonga,Stenotaphrum secundatum,Paspalum
dilatatum,Lolium perenne,Lotus tenuis)withwidegrass
availability and ad libitum water consumption.
Groups and Treatments
Calves were assigned to one of four homogeneous groups
according to weight, sex, and age (n= 10 each group), and
treated as follows: Cu group, 0.3 mg/kg Cu edetate; Zn group,
1 mg/kg Zn edetate; Cu + Zn group, the same doses of Cu and
Zn edetate (Suplenut®, Biogénesis Bagó); and control group,
saline sterile solution. The animals were subcutaneously
injected every 40 days from November 2015 to March 2016
on days 0, 40, 80, and 120 days.
Blood samples were obtained via jugular venipuncture and
collected in Na
2
EDTA-containing tubes previously washed
with deionized water. They were kept at 4 °C until processing
within 6 h after collection. Blood was centrifuged at 1500 rpm
for 10 min and plasma was proportionally deproteinized with
10% trichloroacetic acid. Copper and Zn concentrations were
measured in supernatants using atomic absorption spectropho-
tometry (AAS) (Perkin Elmer AAnalyst 200). Blood samples
(n= 5 per group) were collected in tubes containing EDTA-K
3
and sent to a regional laboratory (Laboratorio Azul SA) to be
processed for evaluation of hematological parameters (red cell
count, hemoglobin concentration, hematocrit percentage,
hematimetric indices, white blood cell count, absolute and
relative leukocyte formula, and platelets count).
At the same time, grass was collected from three sites in the
paddock according to animal behavior and forage intake.
Samples were washed, dried, and exposed to acid digestion
(3:1 nitric-perchloric acid mixture). The concentrations of Cu,
Zn, and iron (Fe) were measured with AAS, whereas molyb-
denum (Mo) and sulfur (S) concentrations were measured
using graphite furnace AAS and Arsenazo III titration
(adapted from Hamm et al. [13]), respectively. In grass sam-
ples, Zn, Cu, Mo, and Fe concentrations were measured.
The quality of bovine drinking water was analyzed in a
sample initially collected from water troughs, the only water
source for the animals.
Individual animal weight was recorded early in the morn-
ing after the animals had fasted for 12 h.
Statistical Design and Analysis
We used a completely randomized statistical design. Data
were analyzed using a mixed model of repeated measures over
time and a 2 × 2 factorial arrangement using SAS statistical
software 9.1. The main factor was parenteral supplementation
with or without Zn or Cu. Supplementation with Zn and/or
Cu, all their possible interactions and time were taken as fixed
variables, whereas the animals represented the random vari-
able. The SLICE option was used for mean separation if sig-
nificant differences were reported for the main variables
(p< 0.05), interactions (p< 0.1), or tendencies (p<0.15).
When only the treatment was significant, mean separation
was done by a protected Fisher’s test using the PDIFF-SAS
option. Associations between plasma Cu and Zn concentra-
tions and weight gain were assessed with correlation analysis
using the same statistical software.
Results
In the Cu group, plasma Cu concentration increased after the
second sampling (Cu × time interaction, p<0.01; Table 1).
We also found Cu × Zn interaction (p=0.09;Table 1), being
plasma Cu concentration higher in the Cu + Zn than in the Cu
group (76.2 vs. 73.4 ± 2.1 μg/dL). In turn, plasma Cu concen-
tration was lower in the Zn than in the control group (46.2 vs.
50.7 ± 2.1 μg/dL). In the case of plasma Zn concentration, it
Mattioli et al.
increased after Zn supplementation either alone or together
with Cu (p=0.02; Table1).
In terms of weight, time differences were observed in the
Zn-treated group, finding higher weight gain after the second
sampling (p<0.02; Table 1). Both Cu and Zn concentration
correlated with weight (Cu: r=0.04, p=0.53; Zn: r=0.25,
p<0.01).
Regarding hematological parameters (erythrocytes, leuko-
cytes, and enzymes), no differences were detected in any of
the four study groups (Table 2).
In grass samples, Zn, Cu, Mo, and Fe concentrations were
19±5, 7.3±1.2, 0.7±0.4, 329±80 ppm dry matter (DM),
respectively, and S concentration was 0.13 ± 0.06% DM. No
significant Cu and Zn contents were detected in drinking
water.
Discussion
Cu deficiency may result in reduced daily weight gains in
calves [3], particularly in cases of severe deficiency [11]. In
the present study, marginal Cu deficiency did not affect either
daily weight gain or hematological parameters. Nevertheless,
our data correlated with previous results of Cu supplementa-
tion at the SRB reporting differences in weight gain in calves
with Cu levels lower than 25 μg/dL [14], which are related to
severe deficiency [15]. Likewise, decreased hemoglobin con-
centrations and low white cell counts were found in heifers
only with Cu concentrations lower than 19 μg/dL [12]. The
marginal Custatus reported in the present trial would be due to
the Cu concentration in grass (7.3 ppm DM), which was lower
than the required 10 ppm DM [16]. Moreover, other grass-
related factors that may lead to Cu deficiency, such as Mo,
Fe, and S, showed a moderate concentration, suggesting that
they did not interfere with Cu absorption [17].
AninterestingfindingrelatedtoCubehaviorwastheCu×Zn
interaction (p= 0.09) in the Cu + Zn group, since supplementation
produced higher Cu concentrations as compared with the Cu
group. Probably, Zn could have promoted metallothionein syn-
thesis in the liver with the combined supplementation [18]. This
protein enhances Cu capture and acts as a liver Cu storage through
which ceruloplasmin is produced to constitute the main determi-
nant of plasma Cu concentration [1,19,20]. Furthermore, Cu
concentrations in the Zn group were lower than in the control
group. Supplementation with Zn could have increased Cu require-
ments since some mechanisms depend on both elements. For
example, Cu-Zn superoxide dismutase (Cu-Zn SOD) is an en-
zyme whose action is related to Zn concentration and also requires
Cu for an adequate activity [21].
The study groups supplemented with Zn either alone or
combined with Cu presented higher weight regardless of
Cu supplementation, probably due to the altered intake
and/or feed conversion produced by Zn deficiency in bo-
vines [2,8]. Studies on experimental animals indicate that
Zn deficiency leads to lower water intake [22], altered
thyroid function [23], IGF-1 signaling failure [24], and
Table 1 Copper (Cu) and zinc (Zn) concentration least square means and live weight of pre-weaning calves in the four study groups
Day Groups SEM pvalue
1
Control Cu Zn Cu + Zn Zn Cu Zn × Cu Zn × T Cu × T
Cu concentration (μg/dL)
0 46 42 44 45 1.5 0.67 < 0.01 0.09 0.96 < 0.01
40 49 88 45 89
80 53 87 49 89
120 55 76 47 81
Zn concentration (μg/dL)
0 84 80 85 81 2.3 0.02 0.12 0.77 0.48 0.98
40 112 101 115 117
80 108 102 115 109
120 92 91 109 100
Weight (kg)
0 92 92 93 92 1.0 0.02 0.73 0.43 < 0.01 0.48
40 124
a
124
a
127
b
126
b
80 145
a
146
a
148
b
149
b
120 172
a
173
a
181
b
177
b
SEM standard error of the mean, Ttime
Different letters in the same row indicate p<0.05
1
There was no Cu × Zn × time interaction for any of the variables
Effects of Copper and Zinc Supplementation on Weight Gain and Hematological Parameters in Pre-weaning Calves
anorexia secondary to the suppression of hypothalamic
neuropeptide Y [25].
The National Research Council recommends 30 ppm DM
of Zn to reach the requirements [16] and suggests that lower
average weight gain could occur with Zn dietary doses of
20 ppm DM [11,26]. These data are in agreement with the
19 ppm DM Zn found in the present trial; Zn concentration
was higher in the Zn groups and correlated with weight gain
(r:0.25;p< 0.01). Although it is agreed that Zn concentration
should be taken as an indicator of Zn status in animals, most of
the trials reporting differences in terms of weight gain in
groups supplemented with or without Zn showed similar Zn
concentrations [27,28]. The time (weeks-months) required for
the development of the deficiency as an indicator of Zn status
might improve Zn concentration. In a previous trial, it was
reported that 6 weeks were needed to distinguish the Zn-
supplemented (40 ppm DM) from the control group
(17 ppm) [29]. Other studies obtained similar results in
3 weeks, i.e., low daily weight gain but no differences in
plasma Zn concentration [8]. In this trial, Zn supplementation
every 40 days during 4 months was associated with higher
weight gain. Further research showing the importance of herd
risk diagnosis based on plasma Zn concentration could con-
tribute to preventing Zn deficiency in calves.
Conclusions
Zinc parenteral supplementation every 40 days improved the
daily weight gain of calves, indicating the risk for Zn deficien-
cy in the SRB area. Marginal Cu concentration did not induce
lower weight gain, and marginal Cu + Zn concentration did
not alter hematological parameters.
Acknowledgements The authors would like to thank A. Di Maggio for
manuscript correction and edition and to Dr. Darío Piacentini for edetate
Cu and Zn solutions.
Author Contributions G.A.M. and L.E.F. conceived and designed the
experiments; D.E.R., and E.M.G. conducted the experiments; A.E.R.
analyzed the data; E.T., G.A.M., and L.E.F. critically wrote and revised
the paper. All authors read and approved the final manuscript.
Funding This study was supported by a grant from the National Program
of Incentives to Teaching and Research, Secretary of University Policies,
Ministry of Education of Argentina (grant no. 11/V204, School of
Veterinary Sciences, National University of La Plata). The sponsor had
no involvement in the study design, collection, analysis, or interpretation
of the data presented in this paper.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no competing
interests.
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Table 2 Least square means for hematological and serological
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3
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120 6978 6746 6880 6752
Hemoglobin (g/dL)
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120 13.18 13.46 13.66 13.28
Hematocrit (%)
040434144
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12042424442
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GGT (U/L)
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Effects of Copper and Zinc Supplementation on Weight Gain and Hematological Parameters in Pre-weaning Calves
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