Acta Scientiae Veterinariae, 2014. 42: 1190.

RESEARCH ARTICLE

ISSN 1679-9216

Pub. 1190

Levels of Cu, Mn, Fe and Zn in Cow Serum and Cow Milk:

Relationship with Trace Elements Contents and Chemical Composition in Milk
Hui Wang1, Zhiqi Liu2, Yongming Liu1, Zhiming Qi1, Shengyi Wang1,
Shixiang Liu1, Shuwei Dong1, Xinchao Xia1 & Shengkun Li1

ABSTRACT

Background: Milk can be considered a source of macro- and micronutrients, together with bioactive substances, and also
contains a number of active compounds that play a significant role in both nutrition and health protection. Data on milk
chemical composition provide information on milk quality alterations and cow health status but is also useful in planning
feeding and breeding. Animals living use changes in the photoperiod to adapt to seasonal changes in the environment. The
composition of milk is of great importance for the dairy industry and there is great interest in changing the composition of
milk. Dairy cow breeding in China has always been restricted by species, management, nutrient supply, seasonal variation
and other factors, which cause the quality of raw milk is unsteady. The transition element cations have concentrations in
blood, tissues and milk that are largely independent of the intake, as they relate to regulation of gut absorption and changing metabolic demands. Thus, the main objective of this study was to investigate the possible effects of nature month and
physiological variables on chemical composition in milk and trace element levels in Chinese Holstein Friesian cows.
Materials, Methods & Results: In this paper, cow milk samples were collected from 180 consecutive milkings during 6
month. For the first time, Pearson phenotypic correlations and hierarchical cluster analysis (HCA) were used for analyzing
milk chemical composition and trace elements levels in milk and serum. Linear regression was used to predict the value
of the continuous dependent variable based on the value of the independent variable. The results showed milk chemical
composition and trace elements vary considerably throughout the test period. HCA classified samples into three main groups
on the basis of the measured parameters. The first group was composed of fat, total solids, Mn, Fe, and Zn; the second
cluster included solids non fat, freezing point, total protein, density, lactose, and acidity; and the last cluster consisted of
Cu alone. Stepwise linear regression analysis showed that milk Mn was significantly correlated with serum Mn (r = 0.388,
P = 0.008 < 0.05), but milk Cu, Fe and Zn levels were not positively associated with serum (r = 0.013, P = 0.933 > 0.05;
r = 0.235, P = 0.087 > 0.05; r = 0.217, P = 0.081 > 0.05, respectively).
Discussion: These observations suggest that the concentration of Cu, Mn, Fe and Zn in serum and milk did not remain
stable during lactation period. Nature month variations have to be taken into consideration for the correct interpretation
of milk chemical composition and trace elements status in cow. HCA is an effective way to comprehensively evaluate the
characterization of milk, which not only could avoid the bias and the instability of single factor analysis, but also reflect the
relationship between different chemical composition and trace elements related characterization and quality in milk better.
The newborn infant is dependent on an adequate supply of trace elements for optimal nutrition and health. The mammary
gland has a remarkable capacity to adapt to maternal deficiency or excess of trace elements and to homeostatically control
milk concentrations of these essential nutrients such as Cu, Mn, Fe and Zn. However, the content of milk Cu, Fe, and Zn
is not suitable for reflect the states of the corresponding nutrients in serum.
Keywords: milk, chemical composition, trace elements, relationship, serum.

Received: 15 December 2013

Accepted: 12 May 2014

Published: 23 May 2014

Engineering and Technology Research Center of Traditional Chinese Veterinary Medicine of Gansu Province, Key Lab of New Animal Drug Protection
of Gansu Province, Key Lab of Veterinary Pharmaceutical Development of Ministry of Agriculture, Lanzhou Institute of Husbandry and Pharmaceutics
Sciences of Chinese Academy of Agricultural Sciences, Lanzhou, Gansu, P.R. China. 2Institute of Agro-Products Processing Science and Technology,
Chinese Academy of Agricultural Sciences, Beijing, P.R. China. CORRESPONDENCE: H. Wang [wanghui01@caas.cn - Tel.: +86 (931) 211-5263].
Lanzhou Institute of Husbandry and Pharmaceutics Sciences of Chinese Academy of Agricultural Sciences. No 335, Qilihe, Lanzhou, 730050, China.

H. Wang, Z. Liu, Y. Liu, et al. 2014. Levels of Cu, Mn, Fe and Zn in Cow Serum and Cow Milk: Relationship with Trace Elements Contents
and Chemical Composition in Milk.
Acta Scientiae Veterinariae. 42: 1190.
INTRODUCTION

of the animals was supervised continuously, and any

animal presenting any sign of disease was removed
from the study. As concerns their feeding, the forage
fraction of the diets was constituted of alfalfa hay and
corn silage. Individual milk samples (1 per animal)
were collected during the morning milking of a test
day. After collection and with no preservative added,
milk samples were stored in portable refrigerators
(4C) and transferred to the milk quality laboratory of
the Lanzhou Institute of Husbandry and Pharmaceutics
Sciences of Chinese Academy of Agricultural Sciences
(Gansu, China).
Milk protein percentage (MPP), milk fat percentage (MFP), total solids percentage (TSP), solids
non fat (SNF), milk lactose percentage (MLP), milk
density (MD), freezing point (FP), and acidity were
obtained using a milk composition analyzer1.

Milk can be considered a source of macro- and

micronutrients, together with bioactive substances, and
also contains a number of active compounds that play a
significant role in both nutrition and health protection
[34]. The main components of milk from cow not only
reflect the hereditary character, milk secretion character and nutritional status of cow, but also reflect the
seasonal changes [29,30]. Animals living use changes
in the photoperiod to adapt to seasonal changes in the
environment [26]. The composition of milk is of great
importance for the dairy industry and there is great
interest in changing the composition of milk. The
scope for increasing concentration of micronutrients
in milk is limited by the complex and co-coordinated
biochemical mechanisms of animal homeostasis [27].
Dairy cow breeding in China has always been restricted
by species, management, nutrient supply, seasonal variation and other factors, which cause the quality of raw
milk is unsteady [41]. Trace elements play a versatile
function in the human body ranging from developing
immunity to provide antioxidant defense [33,43,44].
The transition element cations have concentrations in
blood, tissues and milk that are largely independent of
the intake, as they relate to regulation of gut absorption
and changing metabolic demands [9,39]. The correlation among chemical composition and trace elements
in milk, and the relationship of trace elements between
milk and serum were few analyzed. Thus, the main
objective of this study was to investigate the possible
effects of nature month and physiological variables on
chemical composition in milk and trace element levels
in Chinese Holstein Friesian cows.

Serum sampling

A total of 180 blood samples were collected

during the experiment. Approximately 10 mL blood
was drawn from each cow by jugular vein puncture, using a plastic syringe fitted with a stainless steel needle.
The blood sample was collected into a metal-free
plastic tube and allowed to clot at room temperature
for half an hour and then centrifuged at 3,000 rpm/min
for 15 min in a dust-free room. Serum samples were
stored at -70C and protected from light until analysis.
Analysis of copper, manganese, iron, and zinc

Milk (0.2 mL) and serum (0.2 mL) samples

were placed in PTFE digestion tubes and 12 mL diacid
mixture (HNO3:HCl; 3:9) was added. The samples
were analyzed after microwave digestion using microwave digestion system2. The optimal operating
condition was developed as described in our previous
publication [38]. The digested samples were cooled
to room temperature, transferred to glass tube, 1 mL
HClO4 was added, and removed the diacid mixture until
1 mL was left using intelligent temperature controller
in 110C. The digested solution was transferred to
volumetric flask, and volume made up to 10 mL with
ultra pure water. A blank digest was carried out in the
same way.
Four elements (Cu, Mn, Fe, Zn) were measured by atomic absorption spectrophotometer (AAS)3
with a deuterium background corrector. The elements
were determined by using airacetylene flame. The
procedure used for measuring concentrations of trace

MATERIALS AND METHODS

Milk sampling and analysis of milk composition

The milk samples analyzed in this study were

obtained from Dingxi Tianchen animal husbandry co.,
LTD, with Chinese Holstein Friesian cows. The farm
is located in Gansu province, northwest China, at latitude N353227 and E10426 33 , at 2010.08 m above
sea level, with a continental Mediterranean climate,
belongs to semi-arid temperate climate region, and a
total of 350-500 mm precipitation per year.
A total of 180 individuals of Chinese Holstein
Friesian cows were milk-sampled during the period
from March 2012 to August 2012. The health condition

H. Wang, Z. Liu, Y. Liu, et al. 2014. Levels of Cu, Mn, Fe and Zn in Cow Serum and Cow Milk: Relationship with Trace Elements Contents
and Chemical Composition in Milk.
Acta Scientiae Veterinariae. 42: 1190.

Hierarchical cluster analysis (HCA) is a statistical technique used to group cases (individuals
or objects) into homogeneous sub-groups based on
Ward method by using of Euclidean distance criterion was performed for grouping of varieties. HCA
calculates the distances (or correlation) between all
samples, and was applied to the standardized data
to investigate similarities between different samples
and sample types [4]. Chemical composition and
trace elements states of milk were analyzed by HCA
of SPSS.

elements has been described previously [1]. Optimization of the instrument was done for higher sensitivity
and lower detection limits. Multi-element calibration
standards were prepared by appropriate dilution of
1000 g/mL single-element standard solutions.
Statistical analyses

The SPSS software package4, was used for

the statistical analyses of data. We performed initial
descriptive statistics, including mean and standard
deviation (SD). Significance of difference between
two groups was evaluated using Students t-test. For
multiple comparisons, one-way analysis of variance
(ANOVA) was used. Pearsons correlation analyses
were carried out among the contents of four trace
elements and eight chemical compositions in milk,
as well as to evaluate the trace elements relationships
between the level of serum and milk. The p-values
were two-tailed, and two significant levels were using
P = 0.05 and 0.01.

RESULTS

Gross composition of milk samples

Changes in gross composition (MPP, MFP,

TSP, SNF, MLP, MD, FP, and Acidity) of Chinese Holstein Friesian cow milk during the lactation period are
shown in Table 1. The line chart of Figure 1 describes
the changes of chemical composition of milk too.

Table 1. Composition of Chinese Holstein Friesian cow milk over 6 months of lactation.

Composition

March

April

May

June

July

August

MPP (%)

3.3660.327a 3.2320.501ab 3.0830.315b 3.0210.364b 2.8890.535c 3.0630.556b

MFP (%)

3.1121.890bc 4.3602.728ab 4.1772.476ab 4.8513.621ab 3.7912.826b 5.4953.018a

TSP (%)

11.9961.811b 13.6552.943a 12.9292.297ab 13.0903.076ab 12.6872.799ab 13.9722.622a

SNF (%)

8.9480.642a 8.7680.731ab 8.7140.363ab 8.6000.568ab 8.4950.699b 8.4360.772b

MLP (%)

4.4420.470ab 4.4010.335ab 4.5080.182a 4.4610.313a 4.4980.276a 4.2500.489b

MD (g/L)

1031.43.807a 1029.44.774a 1029.83.193a 1029.35.171ab 1029.25.281ab 1026.64.871b

FP (C)

0.5440.041 0.5360.029 0.5320.020 0.5290.026 0.5260.031 0.5290.042

Acidity (T) 7.5520.939a 7.0501.122ab 6.8820.841abc 6.7471.142bcd 6.1061.420d 6.2061.418cd

ad

Means within a row with different superscripts differ (P < 0.05). MPP, milk protein percentage; MFP, milk fat percentage; TSP, total
solids percentage; SNF, solids non fat; MLP, milk lactose percentage; MD, milk density; FP, freezing point.

Normally, fat makes up from 3.5 to 6.0% of

milk, varying between breeds of cattle. The majority
of milk fat is in the form of triglycerides formed by the
linking of glycerol and fatty acids. Milk fat contains
predominantly short-chain fatty acids built from acetic
acid units derived from fermentation in the rumen. The
fat content of milk was instability during the experiment.
Although its range from 3.112 to 4.851% during the
months of March to July, the values were not significant

Milk protein is an important functional nutrient

in bovine milk. Its content is also the capital parameter
for assessing the quality of milk. The concentration of
protein in milk varies from 3.0 to 4.0%. The percentage
varies with the breed of the cow. There was a continuous reduction in protein content (MPP) from 3.366 to
2.889% between the months of March to July. And the
value of MPP in July was lowest compared with other
months (P < 0.05). It had a slight increase in August.

H. Wang, Z. Liu, Y. Liu, et al. 2014. Levels of Cu, Mn, Fe and Zn in Cow Serum and Cow Milk: Relationship with Trace Elements Contents
and Chemical Composition in Milk.
Acta Scientiae Veterinariae. 42: 1190.

In general, the freezing point (FP) is constant

and is influenced by some factors such as variations in
the growth medium, system growth, race, etc. FP was
stability in the range of -0.53 to -0.54C. There was no
significant difference among each month.
Milk density (MD) was stability in the months
of March to July. It was slightly lower than other
months (P < 0.05).
There was a sharp decline in Acidity from
7.552 to 7.050 within the first month. It stabilized during April to June. It continued decreasing gradually to
reach 6.106 at July, and further slightly increased to
6.206 at August.

(P > 0.05). But the value reached 5.495% in August, and

significantly higher than it in March and July (P < 0.05).
Total solids percentage (TSP) of milk was basic
stability in the range of 11.996 to 13.090%, except the
months of April and August, which were a little higher
than other months.
There was a decline in solids non fat (SNF)
from 8.948 to 8.600% during the months of March to
June. It continued decreasing gradually to reach 8.495
and 8.436% at July and August, respectively.
The principal carbohydrate in milk is lactose.
Lactose is one of the components of milk which is
synthesized in mammary gland and therefore will
undergo changes when synthesis is impaired function.
The concentration of lactose in the milk (MLP) is
relatively constant and averages about 4.4%. Lactose
concentration is similar in all dairy breeds and cannot
be altered easily by feeding practices. The lactose
content of milk fell below 4% may indicate an inflammation of the mammary gland.

Changes in the trace mineral content of the milk during

the lactation period

To investigate the changes in the trace mineral

content of the milk during the lactation period, the content of Cu, Mn, Fe and Zn in milk was detected using
AAS. The results were shown in Figure 2.

Figure 1. Changes in gross composition of Chinese Holstein Friesian cow milk during the lactation period. Milk was collected from 180 Chinese Holstein
Friesian cows during the period from March 2012 to August 2012. The data were expressed as the mean standard deviation (SD). MPP, milk protein
percentage; MFP, milk fat percentage; TSP, total solids percentage; SNF, solids non fat; MLP, milk lactose percentage; MD, milk density; FP, freezing point.

H. Wang, Z. Liu, Y. Liu, et al. 2014. Levels of Cu, Mn, Fe and Zn in Cow Serum and Cow Milk: Relationship with Trace Elements Contents
and Chemical Composition in Milk.
Acta Scientiae Veterinariae. 42: 1190.

Figure 2. Amount of Cu, Mn, Fe and Zn in cow milk at different stages of lactation. The content of Cu, Mn, Fe and Zn in cow milk was detected
using AAS. The data were expressed as the mean standard deviation (SD) and statistically analyzed by ANOVA; **Significant at 0.01 probability
level, *Significant at 0.05 probability level.

Copper

Zinc

The mean concentration of Cu in cow milk was

1.167 0.622 g/mL at April, 0.610 0.522 g/mL at
June, and 0.451 0.183 g/mL at August. The results
indicated a significant decline in the mean concentration
of copper during the lactation period of June and August
(Figure 2, P < 0.01).

The mean concentration of Zn was 4.563

0.715 g/mL at April, 4.357 0.697 g/mL at June,
and 4.554 0.899 g/mL at August. The concentration of zinc at June was a little declined, but this
change was not statistically significant (Figure 2, P
> 0.05).

Manganese

Changes in the trace mineral content of the serum during

the lactation period

The mean concentration of Mn was 0.714

0.049 g/mL at April, 0.806 0.052 g/mL at June, and
0.817 0.104 g/mL at August. The mean concentration increased slightly from April to June, and it was
statistically significant (Figure 2, P < 0.01).

Copper

The mean concentration of Cu in cow serum was 0.365 0.089 g/mL at March, 0.421
0.103 g/mL at April, 0.432 0.126 g/mL at May,
0.429 0.147 g/mL at June, 0.740 0.175 g/
mL at July, and 0.680 0.107 g/mL at August.
The results indicated a significant increase in the
mean concentration of copper during the lactation
period of July and August in cow serum (Figure
3, P < 0.01).

Iron

The mean concentration of Fe was 5.789

4.590 g/mL at April, 8.57 2.87 g/mL at June, and
3.326 0.867 g/mL at August. The mean concentration increased rapidly from April to June, and it was
statistically significant compared with August (Figure
2, P < 0.01).

H. Wang, Z. Liu, Y. Liu, et al. 2014. Levels of Cu, Mn, Fe and Zn in Cow Serum and Cow Milk: Relationship with Trace Elements Contents
and Chemical Composition in Milk.
Acta Scientiae Veterinariae. 42: 1190.

Figure 3. Amount of Cu, Mn, Fe and Zn in cow serum at different stages of lactation. The serum Cu, Mn, Fe and Zn levels in cow were measured
using AAS. The data were expressed as the mean standard deviation (SD) and statistically analyzed by ANOVA; **Significant at 0.01 probability
level, *Significant at 0.05 probability level.

Manganese

Zinc

The mean concentration of Mn was 0.665

0.169 g/mL at March, 0.873 0.356 g/mL at April,
1.015 0.074 g/mL at May, 1.313 0.077 g/mL at
June, 0.540 0.238 g/mL at July, and 0.482 0.119
g/mL at August. There was a sharp increase from
0.665 to 1.313 g/mL during the months of April to
June (P < 0.01 or P < 0.05). It was sharp declined between June to July (P < 0.01). Last it stabilized during
July to August (Figure 3).

The mean concentration of Zn was 1.641

0.313 g/mL at March, 1.874 0.353 g/mL at April,
2.044 0.604 g/mL at May, 1.925 0.377 g/mL at
June, 1.638 0.218 g/mL at July, and 2.248 0.551
g/mL at August. The concentration of zinc increased
gradually to reach 2.044 g/mL at May (P < 0.01), then
declined to 1.638 g/mL at July, and further increased
to 2.248 g/mL at August (Figure 3, P < 0.01).

Iron

Correlations among the contents of chemical composition

and four trace elements in milk

The mean concentration of Fe was 3.440

1.514 g/mL at March, 4.381 1.955 g/mL at April,
3.128 1.878 g/mL at May, 3.700 1.659 g/mL at
June, 4.896 1.274 g/mL at July, and 5.321 0.941
g/mL at August. The mean concentration increased
rapidly from March to April (P < 0.05), then declined
from 4.381 to 3.128 g/mL between April to May. It
stabilized during May to June. It continued increasing
gradually to reach 4.896 g/mL at July, and further
slightly increased to 5.321 g/mL at August (Figure
3, P < 0.01).

To investigate the relationships among chemical composition and four trace element contents in
milk, Pearson correlation analyses were performed
for the accessions (Table 2). Among the chemical
composition and trace elements, closely positive associations were recognized between the contents of
MPP and SNF or MD or FP or Acidity or Cu; between
the contents of MFP and TSP or Zn; between the
contents of TSP and FP; between the contents of SNF
and MLP or MD or FP or Acidity or Cu; between the
contents of MLP and MD or FP or Acidity; between

H. Wang, Z. Liu, Y. Liu, et al. 2014. Levels of Cu, Mn, Fe and Zn in Cow Serum and Cow Milk: Relationship with Trace Elements Contents
and Chemical Composition in Milk.
Acta Scientiae Veterinariae. 42: 1190.

between TSP and SNF or MLP or MD or Acidity; or

between Acidity and Fe were indicated that there were
very significantly negative associations (P < 0.01),
and visibly positive correlations were recognized
between MLP and Fe; between Acidity and Fe (P <
0.05) [Table 2].
These results suggested that high Cu content
might be accompanied with high MPP and SNF contents of milk; high Zn content might be accompanied
with high MFP.

the contents of MD and FP or Acidity; or between the

contents of FP and Acidity or Cu (P < 0.01).
There were significantly positive correlations between the contents of MFP and Fe; between
the contents of MD and Cu; between the contents of
Acidity and Cu; or between the contents of Fe and Zn
(P < 0.05), whereas no close correlations were found
between Mn content and the other chemical composition or the other mineral elements (P > 0.05).
The relationships between MPP and MFP or
TSP; between MFP and SNF or MLP, MD or Acidity;

Table 2. Pearson phenotypic correlations among milk chemical composition and milk trace elements.

MFP
MPP

TSP

0.432** 0.328**

SNF

MLP

MD

FP

Acidity

Cu

Mn

Fe

Zn

0.820**

0.057

0.551**

0.604**

0.856**

0.419**

0.123

0.037

0.123

0.094

0.516**

0.122

0.229

0.399*

0.422**

MFP

0.930** 0.515** 0.251** 0.836**

TSP

0.431** 0.291** 0.814**

0.045

0.455**

0.225

0.219

0.285

0.119

SNF

0.525**

0.823**

0.796**

0.795**

0.390**

0.052

0.171

0.175

0.631**

0.476**

0.349**

0.027

0.086

0.329*

0.107

0.695**

0.319*

0.126

0.384*

0.231

0.544**

0.363*

0.028

0.060

0.180

0.347*

0.195

0.419**

0.244

0.284

0.070

0.003

0.308

0.048

MLP
MD

0.422**

FP
Acidity
Cu
Mn

Fe
0.309*
**Significant at 0.01 probability level. *Significant at 0.05 probability level. MPP, milk protein percentage; MFP, milk fat percentage;
TSP, total solids percentage; SNF, solids non fat; MLP, milk lactose percentage; MD, milk density; FP, freezing point.

Hierarchical cluster analysis (HCA)

tissues of humans and animals. Cu, Zn, Mn and Fe are

found as key components in a multitude of enzymes
and play an important role in many physiological functions of humans and animals.
Linear regression as a statistical procedure
was used to predict the value of the continuous dependent variable (Milk Cu) based on the value of the
independent variable (Serum Cu). This study found no
significant correlation between serum Cu and milk Cu
in their concentration values for dairy cows (r = 0.013,
P = 0.933 > 0.05) [Figure 5A]. Manganese (Mn) is an
essential mineral nutrient in humans and other animals
and is required for normal amino acid, lipid, protein,
and carbohydrate metabolism [40]. In this study, stepwise linear regression indicated that milk Mn levels
were positively associated with serum Mn concentrations (r = 0.388, P = 0.008 < 0.05) [Figure 5B].
Iron is a part of both the oxygen-carrying system
and ironsulfur proteins, which play an important role
in oxygen and carbon dioxide transport in vertebrates

With the hierarchical cluster analysis three clusters have been distinguished for the well data. The result of
cluster analysis as a tree diagram was shown in Figure 4,
in which three well-defined clusters were visible. Samples
were grouped in clusters in terms of their nearness or
similarity. Cluster analysis (CA) used less information
(distances only). It was interesting to observe what kind of
classification can be made on the basis of distances only.
The first group of samples (A) was clearly discernible
which was composed of MFP, TSP, Mn, Fe, and Zn. The
second cluster (B) included SNF, FP, MLP, MD, MPP,
and Acidity, and the last cluster (C) consisted of Cu alone.
The tendencies to form natural sample groupings arising
from common analytical characteristics were clear in this
data analysis procedure.
Serum trace elements status in relation to milk trace elements status

Copper, zinc, iron, and many dietary essential

elements are desired trace constituents of the healthy

H. Wang, Z. Liu, Y. Liu, et al. 2014. Levels of Cu, Mn, Fe and Zn in Cow Serum and Cow Milk: Relationship with Trace Elements Contents
and Chemical Composition in Milk.
Acta Scientiae Veterinariae. 42: 1190.

and in the electron transport system of mitochondria,

respectively. The date in this study demonstrated a negative correlation between milk iron and serum iron in their
concentration values for cows, but did not reached significant level (r = -0.235, P = 0.087 > 0.05; Figure 5C).
The correlation of Zn statistically no significant evidence

was found between milk and serum in their concentration

values for cows (r = 0.217, P = 0.081 > 0.05; Figure 5D).
DISCUSSION

Milk plays a tremendous role in building a

healthy society and can be used as vehicle for rural de-

Figure 4. Dendrogram of hierarchical cluster analysis of milk chemical composition and milk
trace elements.

Figure 5. Relationship between serum trace elements and milk trace elements. (A) Correlation between serum Cu
and milk Cu (r = 0.013, P = 0.933). (B) Correlation between serum Mn and milk Mn (r = 0.388, P = 0.008). (C)
Correlation between serum Fe and milk Fe (r = 0.235, P = 0.087). (D) Correlation between serum Zn and milk Zn
(r = 0.217, P = 0.081).

H. Wang, Z. Liu, Y. Liu, et al. 2014. Levels of Cu, Mn, Fe and Zn in Cow Serum and Cow Milk: Relationship with Trace Elements Contents
and Chemical Composition in Milk.
Acta Scientiae Veterinariae. 42: 1190.

velopment, employment and slowing down the migration of the rural population. The milk composition has
changed during the past decades because of changes
in the feeding regimen and breeding practices or other
changes in dairy husbandry. The main objective of this
study was to investigate the possible effects of nature
month and physiological variables on composition of
milk and trace element levels in cow, and the relationship between trace element contents and chemical
composition in milk.

Trace metal levels are important for human

health. Milk is an excellent source of most minerals
required for the growth of the young. Cu, Mn, Fe and
Zn can be considered trace minerals with a central role
in many metabolic processes throughout the body and
are essential for correct growth and development of all
animals. They predominantly act as catalysts in many
enzyme and hormone systems which influence on
growth, bone development, feathering, enzyme structure and function, and appetite [31,35]. Fe is essential
for maintaining proper cell functions and is normally
tightly controlled by transporter and storage proteins
[22]. The low Fe concentration in milk cannot meet the
needs of the young, but this low level turns out to have
a positive aspect because it limits bacterial growth in
milkiron is essential for the growth of many bacteria
[3]. Zn is acting as a catalytic, structural, and regulatory
ion. Moreover, Zn-binding protein (metallothionein)
plays a key role in Zn-related cell homeostasis which,
in turn, is relevant also against oxidative stress, including exposure to oxyradicals, inflammation, infection,
and immune responses [36].
The results of this research found that the concentration of Cu, Mn, Fe and Zn in serum and milk did
not remain stable during lactation period. Our finding
is not similar with Erdogan et al. reported that seasonal
changes had no significant effect on manganese, copper, zinc, and iron concentrations on cows and their
milk [7].

Nature month variation of chemical composition in milk

A systematic study on describes the variations milk composition is of foremost importance

to evaluate the milk production ability of a milking
animal. The content of TSP (11.996 to 13.972%),
MFP (3.112 to 5.495%), MPP (2.889 to 3.366%) and
SNF (8.436 to 8.948%) in Chinese Holstein Friesian
cows milk during lactation stages was found to
be lower than that of sheep (17.48 to19.50, 8.0 to
9.6, 5.32 to 7.74 and 9.48 to 10.1%), goat (12.60 to
15.17, 3.9 to 5.7%, 3.3 to 3.7 and 8.53 to 9.47%),
and buffalo (12.73 to 15.90%, 4.0 to 6.5%, 3.12 to
4.12% and 8.28 to 9.40%) [16]. The content of MLP
(4.250 to 4.508%) was found to be comparable with
that of buffalo (3.28 to 4.80%), cow (3.0 to 4.6%),
goat (4.0 to 5.5%) and sheep (3.0 to 4.2%) [16]. In
the present study, lactose content did not vary significantly throughout the lactation. Lactose is the
main determinant of milk volume. A close relationship between lactose synthesis and the amount of
water drawn into milk makes lactose a stable milk
component [28].
Of the main milk components (lactose, fat, and
protein), lactose had the smallest and fat the highest
variation, with protein in between. This is in line with
the general observation that fat is the most sensitive
component of milk to dietary changes and lactose is
the least sensitive, again with protein in between [14].
The variation trend is consistent with the literature
reported [12]. The milk composition within the same
animal breed not only depends on the state of lactation, but also on genetic factors, nutritional status of
the animals as well as the composition of feed and the
environment [18].

Characterization of chemical composition and trace elements in milk based on genetic and herd parameters

Hierarchical cluster analysis (HCA) is an exploratory tool designed to reveal natural groupings (or
clusters) within a data set that would otherwise not be
apparent, and it is a method for placing objects into
more or less homogeneous groups so that the relation
between the groups is revealed [13]. It is most useful
when you want to cluster a small number (less than
a few hundred) of objects. In HCA, samples were
grouped on the basis of similarities, without taking into
account the information about the class membership.
Cluster analysis is a powerful exploratory technique,
its results must be considered within the context of
the selected input variables and the requirement of an
adequate ratio between the sample size and the number
of variables to be modeled [24].
However, thus far, there have been no reports
using cluster analysis to comprehensively analyze

Changes in the trace elements content of the milk and

serum during the lactation period

H. Wang, Z. Liu, Y. Liu, et al. 2014. Levels of Cu, Mn, Fe and Zn in Cow Serum and Cow Milk: Relationship with Trace Elements Contents
and Chemical Composition in Milk.
Acta Scientiae Veterinariae. 42: 1190.

not only could avoid the bias and the instability of

single factor analysis, but also reflect the relationship between different chemical composition and
trace elements related characterization and quality
in milk better.

chemical composition and trace elements in milk from

cows. A cluster analysis was performed to visualize
the chemical composition and trace elements in milk
correlations in this study. It is clearly shown that three
clusters have been distinguished for the well data. It
is an effective way to comprehensively evaluate the
characterization of milk by cluster analysis which

Milk trace elements status in relation to serum trace ele-

Figure 6. Model structure for trace elements transfer into milk. Trace elements (TE) present either as free (TEf) or bound (TEb) in the body. The letter
B represents TE-binding capacity in the tissue. TE in diet were digested and absorbed in small intestine, and were inhaled into lung, respiratory nose
and olfactory nose, then entered mammary gland, bone, liver, etc, through the circulation. Milk synthesis starts in the epithelial cells of the mammary
gland at the end of pregnancy to support the nutrition and promote the health of the off-spring. Cu was transferred by copper transporters-1 (Ctr1), ATP7A,
and ATP7B, Mn was transferred by transmmlganin (Tm), Fe was transferred by transferrin receptor (TR), divalent metal ion transporter 1 (DMT1), and
ferroportin (Fp), and Zn was transferred by zinc import protein (Zip3) and zinc transporters (ZnT-1, ZnT-2, and ZnT-4) from mammary gland to milk
[6,20,21,23,42]. In our research, the correlation of copper, manganese, iron and zinc between serum and milk was 0.013 (CuSr), 0.388 (MnSr), 0.235
(FeSr) and 0.217 (ZnSr), respectively. With the hierarchical cluster analysis (HCA) 3 clusters have been distinguished the trace elements contents and
chemical composition in milk, and (A) was composed of MFP, TSP, Mn, Fe, and Zn, (B) included SNF, FP, MLP, MD, MPP, and Acidity, and the last
cluster (C) consisted of Cu alone, respectively.

ments status

against hydrogen peroxide and superoxide anion involved in pathogens of cardiovascular disorders by
SOD as antioxidant enzymes [10,15]. Previous studies
have shown that Cu plays an important role in lactation performance in dairy cows [37]. Mammary gland
copper metabolism is regulated by copper transporter
1 (Ctr1), ATP7A, and ATP7B [23] (Figure 6). Kelleher and Lnnerdal used the rat as a model to study
mechanisms regulating milk Cu levels during lactation [17]. The possible mechanisms that the decrease

The newborn infant is dependent on an adequate supply of trace elements for optimal nutrition
and health. The mammary gland is capable of regulating concentrations of essential elements such as Cu,
Mn, Fe and Zn in milk to protect the newborn infant
against deficiency and excess of these elements [23].
Cu is an essential trace element that participates in the pathogenesis of numerous heart diseases
[19]. Cu has a protective impact on cell membranes

10

H. Wang, Z. Liu, Y. Liu, et al. 2014. Levels of Cu, Mn, Fe and Zn in Cow Serum and Cow Milk: Relationship with Trace Elements Contents
and Chemical Composition in Milk.
Acta Scientiae Veterinariae. 42: 1190.

a catalytic or structural cofactor for many different

proteins [6]. Zinc-dependent proteins are found in the
nucleus, the endoplasmic reticulum, Golgi, secretory
vesicles, and mitochondria. Zn homeostasis is complex,
involving both Zn import protein (Zip3) and Zn transporters (ZnT-1, ZnT-2, and ZnT-4) [6,23] (Figure 5D
and Figure 6). Milk Zn concentrations are considerably
higher than in serum. Previous studies found no significant correlation between milk Zn concentration and
serum Zn concentration or maternal dietary Zn intake
[11,25]. The correlation of Zn was found no statistically
significant evidence between milk and serum in their
concentration values for cows (r = 0.217, P = 0.081 >
0.05; Figure 5D).

in serum Cu that occurs during lactation is partially

responsible for decreasing milk Cu levels by reducing
the supply of Cu to the mammary gland. Lnnerdal
reported that the concentration of Cu in human milk
is about 20-25% of that in serum [23]. But in our
study, we found the concentration of Cu in milk was
0.4511.167 g/mL, in serum was 0.3650.740 g/
mL. And the correlation between serum Cu and milk
Cu was not significant (r = 0.013, P = 0.933 > 0.05)
[Figure 5A and Figure 6].Mn is an essential mineral
nutrient in humans and other animals, and is required
for normal amino acid, lipid, protein, and carbohydrate metabolism [40]. Mn present in all tissues is
crucial for immune function, regulation of cellular
energy, antioxidant enzymes, reproduction, digestion,
bone growth, and blood clotting [2,8,42]. In mammalian tissues, Mn is found in only three important
oxidation states: II, III and IV. Free Mn concentrations in plasma/serum. Mn has a tendency to bind to
high molecular mass proteins, such as transmanganin,
transferrin (Tf), albumin and 2-macroglobulin [42]
(Figure 6). In our study, stepwise linear regression
indicated that milk Mn levels were positively associated with serum Mn concentrations (r = 0.388, P =
0.008 < 0.01) [Figure 5B]. This is in agreement with
the knowledge that Mn content in human milk has
been positively correlated with increased amount of
Mn intake in lactating women [5].
Fe in serum is virtually exclusively bound
to transferrin (Tf), and tissue Fe uptake is usually
mediated by cellular transferrin receptors (TfRs). No
correlation between milk Fe and mammary gland TfR
expression has been found in animal models [32],
which strongly suggests that regulation of milk Fe concentrations occurs after uptake of Fe by the mammary
gland. Lnnerdal reported the milk Fe concentration
is ~20-30% of serum Fe in human [23]; and Zhang
et al. reported the concentration of Fe in mouse milk
is approximately 3 times that of the serum [45]. But
in our research, the concentration of Fe in milk was
3.326-8.570 g/mL, in serum was 3.128-4.896 g/mL.
And there was no statistically significant correlation
between milk Fe status and serum Fe concentration
observed in this study (r = -0.235, P = 0.087 > 0.05)
[Figure 5C and Figure 6].
Zn is an essential nutrient for carbohydrate
metabolism, physiological processes, and many other
biochemical reactions because this metal serves as

CONCLUSION

In conclusion, Nature month variations have to

be taken into consideration for the correct interpretation
of milk chemical composition and trace elements status
in cow. It is an effective way to comprehensively evaluate the characterization of milk by cluster analysis. The
mammary gland has a remarkable capacity to adapt to
maternal deficiency or excess of Fe, Cu, Mn and Zn
and to homeostatically control milk concentrations of
these essential nutrients. However, the content of milk
Cu, Fe, and Zn is not suitable for reflect the states of
the corresponding nutrients in serum. Our method, and
of course other improved and enhanced methods, could
be applied to construct a more comprehensive model
or technology of animal behavior, and could be better
serviced for people.
SOURCES AND MANUFACTURERS
1

Foss MilkoScanTM FT 120, Denmark.

MARS5, CEM Company, USA.

ZEEnit 700, Analytik Jena, Germany.

Version 17.0, SPSS Inc., Chicago, USA.

Acknowledgements. The financial supports from the Central

Public-interest Scientific Institution Basal Research Fund (NO.
1610322013003), National Key Technology Research and Development Program of the Ministry of Science and Technology
of China (NO. 2012BAD12B03), and Special Fund for Agroscientific Research in the Public Interest (NO. 201303040-17)
are greatly appreciated.
Ethical approval. All animal protocols have been reviewed and
approved by the Institutional Animal Care and Use Committee
of Lanzhou Institute of Husbandry and Pharmaceutics Sciences
of Chinese Academy of Agricultural Sciences (Animal use
permit: SCXK20009-0010).

11

H. Wang, Z. Liu, Y. Liu, et al. 2014. Levels of Cu, Mn, Fe and Zn in Cow Serum and Cow Milk: Relationship with Trace Elements Contents
and Chemical Composition in Milk.
Acta Scientiae Veterinariae. 42: 1190.
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