Batistel et al. Journal of Animal Science and Biotechnology (2017) 8:33
DOI 10.1186/s40104-017-0163-7
RESEARCH
Open Access
Peripheral leukocyte and endometrium
molecular biomarkers of inflammation and
oxidative stress are altered in peripartal
dairy cows supplemented with Zn, Mn, and
Cu from amino acid complexes and Co
from Co glucoheptonate
Fernanda Batistel1, Johan S. Osorio2, Muhammad Rizwan Tariq1,3, Cong Li4, Jessica Caputo1, Michael T. Socha5
and Juan J. Loor1*
Abstract
Background: Immune dysfunction and a higher risk of uterine infections are characteristics of the transition into
lactation in dairy cows. The supply of complexed trace minerals, which are more bioavailable, could help overcome
the greater needs of these nutrients in tissues around parturition and early lactation.
Results: Twenty Holstein cows received an oral bolus with a mix of inorganic trace minerals (INO) or complexed
trace minerals (AAC) to achieve 75, 65, 11, and 1 ppm supplemental Zn, Mn, Cu, and Co, respectively, in the
total diet dry matter from -30 d through +30 d relative to parturition. Blood for polymorphonuclear leukocyte
(PMNL) isolation was collected at -30, -15, +10, and + 30 d relative to parturition, whereas endometrium biopsies
were performed at +14 and +30 d. Feeding AAC led to greater PMNL expression of genes related with inflammation
response (DDX58), oxidative stress response (MPO), eicosanoid metabolism (PLA2G4A and ALOX5AP), transcription
regulation (PPARG), and cellular adhesion (TLN1). The upregulation by AAC in endometrium of genes related with
inflammation response (TLR2, TLR4, NFKB1, TNF, IL6, IL1B, IL10, IL8), prostaglandin synthesis (PTGS2, PTGES), and
antioxidant responses (NFE2L2, SOD1) indicated a faster remodeling of uterine tissue and potentially greater
capacity to control a local bacterial invasion.
Conclusions: Data indicate that trace mineral supplementation from amino acid complexes improves PMNL
activity and allows the prompt recovery of uterine tissue during early lactation. As such, the benefits of complexed
trace minerals extend beyond an improvement of liver function and productive performance.
Keywords: Inflammation, Oxidative stress, Trace minerals, Transition period
* Correspondence: jloor@illinois.edu
1
Department of Animal Sciences and Division of Nutritional Sciences,
University of Illinois, 1207 West Gregory Drive, Urbana, IL 61801, USA
Full list of author information is available at the end of the article
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Batistel et al. Journal of Animal Science and Biotechnology (2017) 8:33
Page 2 of 12
Background
The transition to lactation is a challenging period for
dairy cows in large part because the immune system,
e.g., neutrophil migration and phagocytosis, is generally
dysfunctional [1–4]. Besides the hormonal and metabolic
changes that contribute to a dysfunctional immune system, during parturition the physical barriers in the cervix, vagina and vulva also are compromised providing
the opportunity for bacteria from the environment as
well as the animal’s skin and feces to ascend the genital
tract, hence, predisposing the cow to uterine diseases
[5]. In the first 2 weeks after calving, 80–100% of cows
present uterine colonization by bacteria, and an optimal
response by the immune system is essential to rapidly
eliminate the pathogens [5].
Neutrophils account for ca. 25% of leukocytes in bovine peripheral blood of healthy animals and they are
the first line of innate immune defense against invading
pathogens [2]. During uterine infection, toll-like receptors on endometrial cells recognize pathogen-associated
molecules, leading to secretion of cytokines, antimicrobial peptides, and chemokines [6]. Chemokines recruit
polymorphonuclear leukocytes (PMNL) into the site of
infection within minutes and promote direct action
against the microbes and attract lymphocytes; however,
persistent infiltration is detrimental because the site of
infection is continually exposed to pro-inflammatory cytokines and reactive oxygen metabolites (ROM) leading
to chronic inflammation and oxidative stress and consequently subclinical endometritis and infertility [6].
Trace minerals are key components of antioxidant systems, metabolic reactions, protein synthesis pathways, and
membrane integrity (physical barrier to pathogens) [4]. In
postpartum dairy cows, supplementation of trace minerals
(e.g., Zn, Se and Cu) benefits the immune system, and
PMNL adhesion and superoxide production [7, 8]. While
the demand of trace minerals increases around parturition, the blood and liver concentrations of trace minerals
decreases [7, 9]. Thus, we hypothesized that supplementation of trace minerals through more bioavailable forms,
e.g., amino acid complexes, would benefit recovery of the
endometrium and the innate immune response at least in
part by altering the expression of genes associated with
PMNL activity and inflammation. Therefore, the objective
of the present study was to evaluate the effects of organic
trace mineral supplementation on expression of key genes
associated with inflammation, oxidative stress, and eicosanoids in PMNL and endometrium tissue. Production responses and biomarkers of energy balance have been
reported elsewhere [10].
Animal Care and Use Committee (IACUC) of the University of Illinois (Protocol #12097).
Methods
All the procedures for this study were conducted in accordance with the protocol approved by the Institutional
Animals, experimental design, and dietary treatments
Details of the experiment design have been published
previously [10]. Briefly, 44 multiparous Holstein cows
were blocked (6 cows per block) according to parity,
previous lactation milk yield, and expected day of parturition. All cows received a common diet from -110 to
-30 d relative to parturition and were supplemented at
100% of the National Research Council [11] requirements with Zn, Mn, Cu, and Co in the form of an inorganic trace mineral mix (INO). From -30 d relative to
expected day of parturition, cows received a common
prepartal diet (close-up diet), and from calving to 30 d
in milk (DIM) a common postpartal diet (fresh diet).
Both close-up and fresh diet were partially supplemented
with an INO mix of Zn, Mn, and Cu to supply 35, 45,
and 6 ppm, respectively, of the total dietary minerals.
The diets and chemical composition are presented in
Table 1. At -30 d relative to parturition, cows were randomly assigned to an oral administration of a bolus once
daily at the time of feeding the TMR. This contained a
mix of either inorganic (INO) or complexed (AAC) Zn,
Mn, Cu, and Co to achieve 75, 65, 11, and 1 ppm supplemental, respectively, in the total diet dry matter intake (DMI). The complexed trace minerals were
provided as Availa®Zn (Zn AA complex), Availa®Mn (Mn
AA complex), Availa®Cu (Cu AA complex), and CoPro®
(Co glucoheptonate) (Zinpro Corp, Eden Prairie, MN)
and the inorganic trace minerals in sulfate form. IACUC
approved uterine biopsies in a maximum of 12 cows per
group, which was deemed appropriate to detect statistical significance based on previous research [12–14].
However, only 20 (AAC = 9; INO = 11) out of 44 cows
used for this study had a complete set of uterine endometrial biopsies and PMNL isolations. Per IACUC
guidelines, cows with a clinical disorder could not continue on experiment; thus, a total of 7 cows had to be
removed from the experiment due to clinical ketosis,
clinical mastitis, retained placenta, displaced abomasum,
or leg fracture [10]. All cows used for PMNL and endometrium gene expression were clinically-healthy.
Sample collection
Blood samples (120 mL) were collected from the tail vein
using 20-gauge BD Vacutainer needles (Becton Dickinson,
Franklin Lakes, NJ) and vacutainers (8 mL, Becton Dickinson, Franklin Lakes, NJ) containing solution A of trisodium citrate, citric acid and dextrose (ACD) at -30, -15,
+10 and +30 d relative to parturition. After blood collection, the tubes were mixed well by inversion and placed
on ice until PMNL isolation (~30 min).
Batistel et al. Journal of Animal Science and Biotechnology (2017) 8:33
Page 3 of 12
Table 1 Ingredient and analyzed chemical composition of diets
fed during close-up (-30 d to calving) and early lactation (1 to
30 d in milk)
Endometrial biopsies were collected by a single individual at +14 and +30 d relative to calving following
similar procedures described previously [15]. Briefly, an
epidural was performed (4 mL of 2% lidocaine) prior to
introducing a Hauptner biopsy instrument protected
with a sanitary chemise into the vagina. Manipulation
per rectum allowed the biopsy tool to pass through the
cervix, after which the biopsy instrument alone was introduced into the uterus subsequent to rupturing the
sanitary chemise at the external cervical orifice. The tool
was guided into the uterine horn approximately 5 cm
past the uterine bifurcation. The tip of the biopsy instrument inside the uterus was carefully identified using the
non-operating hand per rectum. This approach should
have allowed the reproducible procurement of tissue.
With the help of the hand in the rectum, the medial uterine wall was gently pressed into the open instrument jaws
prior to closing the jaws and withdrawing the instrument.
No attempt was made to determine the relative contribution of caruncular and non-caruncular tissue in the biopsies, even though there is some evidence for differences in
transcriptome profiles [16]. The tissue clipped off was immediately placed in liquid nitrogen and frozen at -80 °C
until RNA extraction.
Componenta
Far-off
Close-up
Early lactation
Alfalfa silage
12.2
7.6
4.9
Alfalfa hay
-
3.5
3.9
Corn silage
33.6
38.9
33.1
Wheat straw
34.8
8.4
2.6
Ingredient, % of DM
Cottonseed
-
-
3.9
Wet brewers grains
-
6.1
9.4
Ground shelled corn
4.9
18.8
22.6
Soy hulls
2.0
4.1
3.9
Soybean meal, 48% CP
8.9
3.0
5.6
Expeller soybean mealb
-
0.7
0.2
SoyChlorc
0.2
2.3
-
Blood meal 85% CP
1.0
0.6
0.3
Molasses
-
0.4
-
0.3
-
0.7
Urea
Rumen-inert fat
d
-
-
2.0
Limestone
0.8
2.2
1.6
Salt (plain)
0.3
-
0.3
Ammonium chloride
-
1.14
-
Dicalcium phosphate
0.1
0.3
0.4
Magnesium oxide
-
0.1
0.1
Magnesium sulfate
0.2
1.4
0.3
Sodium bicarbonate
-
-
0.7
Calcium sulfate
-
-
0.1
Mineral-vitamin mixe
0.2
0.2
0.2
Vitamin Af
0.02
0.03
0.04
Vitamin Dg
0.01
0.02
0.02
h
0.36
0.36
0.20
NEL, Mcal/kg DM
1.25
1.59
1.67
CP, % DM
14.4
14.3
18.7
Vitamin E
Chemical analysis
a
NDF, % DM
53.0
39.1
35.9
ADF, % DM
34.5
23.9
22.2
Zn, mg/kg of DM
103
83
69
Mn, mg/kg of DM
84
76
70
Cu, mg/kg of DM
15.5
14.4
12.3
CO, mg/kg of DM
0.83
0.72
0.19
Basal close up and lactation diets were considered as basal diet plus
inorganic trace minerals, or basal diet plus organic trace minerals
b
SoyPLUS (West Central Soy, Ralston, IA)
c
SoyChlor (West Central Soy)
d
Energy Booster 100 (MSC, Carpentersville, IL)
e
Contained a minimum of 4.3% Mg, 8% S, 6.1% K, 2.0% Fe, 3.0% Zn,
3.0% Mn, 5,000 mg/kg of Cu, 250 mg/kg of I, 40 mg/kg of Co, 150
mg/kg of Se, 2,200 kIU/kg of vitamin A, 660 kIU/kg of vitamin D3,
and 7,700 IU/kg of vitamin E
f
Contained 30,000 kIU/kg
g
Contained 5,009 kIU/kg
h
Contained 44,000 IU/kg
Polymorphonuclear leukocyte (PMNL) isolation and
viability analysis
Complete details of PMNL isolation and viability analysis
are included in the Additional file 1. Briefly, PMNL were
isolated from whole blood collected in ACD-containing
vacutainers. An aliquot (20 μL) obtained during the isolation process was used for PMNL quantification and viability using a granulocyte primary antibody (CH138A,
Veterinary Microbiology and Pathology, Washington State
University, Pullman, WA) followed by a second antibody
(Goat Anti-Mouse IgM, Human ads-PE, Southern Biotech, Birmingham, AL). Cells were fixed with 150 μL of
4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) and
preserved at 4 °C until flow cytometry reading (LSR II,
Becton Dickinson, San Jose, CA). All samples harvested
and used for analysis contained more than 80% PMNL
and had at least 90% viability.
RNA extraction, primer design and evaluation, and
quantitative PCR
Methods for RNA extraction from PMNL and endometrium, primer design and evaluation, cDNA synthesis,
quantitative reverse transcription PCR and gene function
are presented in the Additional files 2, 3 and 4. Briefly,
RNA samples were extracted using Qiazol reagent in
combination with the miRNeasy® Mini Kit (Cat. #217004,
Qiagen). Thirty-two target genes involved in inflammation
response, oxidative stress, eicosanoid metabolism, cellular
receptors, transcription regulation and glucose metabolism
Batistel et al. Journal of Animal Science and Biotechnology (2017) 8:33
Page 4 of 12
were evaluated in the PMNL, while 30 target genes related
to inflammation, oxidative stress, eicosanoid metabolism,
transcription regulation and antimicrobial peptides were
assessed in the endometrium. Primers were designed via
Primer Express 3.0.1 software (Applied Biosystems). Quantitative PCR (qPCR) was performed in an ABI Prism 7900
HT SDS instrument (Applied Biosystems). Details of primer sequences and amplicon size, primer product sequencing information, and qPCR performance are presented in
the Additional file 5, 6, 7 and 8. For PMNL, the internal
controls were GOLGA5, SMUG1, and OSBPL2 [17, 18],
while for endometrium were GAPDH, RPS9, and UXT.
The geometric mean of the internal control genes was
used to normalize the expression data.
Table 2 Effects of supplementing cows with inorganic (INO, n =
11) or complexed (AAC, n = 9) trace minerals during the peripartal
period on mRNA expression (fold-change relative to -30 d
prepartum) of genes related with inflammation response,
oxidative stress, eicosanoids, transcription factors, receptors
and glucose metabolism in polymorphonuclear leukocytes (PMNL)
Statistical analysis
Data were analyzed using the MIXED procedure of SAS
9.3 (SAS Institute Inc., Cary, NC) according to the following model:
Y ijkl ¼
μþDi þ bj þ ck þ T l þ DT il þ eijkl
Where Yijkl represent the dependent variable; μ is the
overall mean; Di is the fixed effect of treatment (i = 1, 2);
bj is the random effect of block (j = 1, …9); ck is the random effect of cow within treatment and block (l = 1…, nij);
Tl is the fixed effect of time (day or week) of the experiment (m = 1,… n); DTil is the fixed effect of treatment by
time interaction; and eijkl is the residual error. Endometrium gene expression results were log2-scale transformed
in order to comply with normal distribution of residuals.
For PMNL, the gene expression data at -15, +10, and +30
d relative to parturition was expressed as fold-change relative to -30 d. Statistical differences were declared significant at P ≤ 0.05 and tendencies at P ≤ 0.10.
Results
PMNL
Inflammation response
The cell surface receptors TLR2 (P = 0.85) and TLR4
(P = 0.48), which are involved in the inflammationresponse were not affected by treatments (Table 1). The
transcription factors STAT3 (P = 0.62), TNF (P = 0.14) and
NFKB1 (P = 0.75) also were not affected by treatments.
Among the proteins that recognize foreign DNA, DDX58
had greater expression (P = 0.05; Table 2 and Fig. 1) in the
AAC compared with INO cows, while there was a tendency
(P = 0.09) for the opposite effect for ZBP1; however, IPS1
(P = 0.23) was not affected by treatments (Table 2). There
was an overall decrease in expression of NFKB1 (P = 0.03)
from -15 to +10 d regardless of treatment (Fig. 1).
Gene
Treatments
SEMa
P value1
Treatment
Time
T × Tb
0.21
0.05
0.01
0.40
1.00
0.13
0.23
0.41
0.22
0.91
0.06
0.75
0.03
0.74
1.17
1.09
0.12
0.62
0.62
0.84
1.27
1.21
0.25
0.85
0.84
0.41
TLR4
1.14
1.43
0.30
0.48
0.11
0.49
TNF
1.33
0.82
0.25
0.14
0.99
0.68
ZBP1
0.97
0.66
0.13
0.09
0.15
0.62
MPO
0.66
0.83
0.18
0.48
<0.01
0.03
NFE2L2
1.32
1.57
0.30
0.54
0.15
0.70
NOX1
0.97
0.99
0.26
0.94
0.61
0.12
S100A8
2.05
1.90
0.40
0.78
0.08
0.98
SOD1
0.73
0.91
0.09
0.14
0.02
0.94
SOD2
1.38
1.72
0.22
0.27
0.70
0.99
SOD3
0.66
0.06
0.31
0.15
0.32
0.70
ALOX5AP
0.98
1.28
0.12
0.09
0.10
0.35
LTA4H
0.71
0.67
0.10
0.76
<0.01
0.18
PLA2G4A
0.84
1.23
0.15
0.06
0.22
0.23
PTGS2
0.93
1.30
0.46
0.53
0.04
0.93
INO
AAC
DDX58
1.02
1.59
IPS1
0.80
NFKB1
0.89
STAT3
TLR2
Inflammation
Oxidative stress
Eicosanoids
Transcription factors
PPARA
1.03
0.63
0.14
0.04
0.01
0.21
PPARD
1.14
1.23
0.16
0.71
0.30
0.74
PPARG
0.84
1.68
0.33
0.09
0.80
0.79
RXRA
1.21
1.03
0.11
0.22
0.05
0.78
ADORA1
1.08
1.55
0.14
0.01
0.08
0.15
ENTPD1
1.14
1.64
0.23
0.12
0.34
0.80
IL10
1.37
1.36
0.20
0.95
0.16
0.85
IL1B
1.22
1.76
0.45
0.38
0.51
0.77
ITGAM
0.75
0.78
0.07
0.74
0.10
0.32
ITGB2
0.99
0.86
0.08
0.22
0.34
0.29
P2RY11
1.02
0.89
0.18
0.60
0.14
0.44
PANX1
0.86
0.95
0.13
0.61
0.02
0.15
SELL
1.41
1.66
0.41
0.65
0.27
0.80
TLN1
0.93
1.04
0.03
0.01
0.60
0.74
VCL
0.74
0.83
0.06
0.25
<0.01
0.76
Receptors
Batistel et al. Journal of Animal Science and Biotechnology (2017) 8:33
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Table 2 Effects of supplementing cows with inorganic (INO, n =
11) or complexed (AAC, n = 9) trace minerals during the peripartal
period on mRNA expression (fold-change relative to -30 d
prepartum) of genes related with inflammation response,
oxidative stress, eicosanoids, transcription factors, receptors
and glucose metabolism in polymorphonuclear leukocytes (PMNL)
(Continued)
AAC treatment (Table 2, Fig. 2). However, the treatments did not alter (all P > 0.10) expression of other receptors measured (SELL, ITGAM, ITGB2, VCL, PANX1,
ENTPD1, P2RY11, IL1B, and IL10) (Table 2, Fig. 2).
Glucose metabolism
LDHA
1.37
0.84
0.20
0.06
0.04
0.95
SLC2A1
0.83
0.56
0.06
<0.01
<0.01
<0.01
1
P values represents the probability of statistical significance for the fixed
effects (treatment, time, treatment × time). Statistical differences were
declared significant at P ≤ 0.05 and tendencies at P ≤ 0.10
a
Largest standard error of the mean is shown
b
Interaction of treatment × time
Oxidative stress
An interaction T × T (treatment × time; P = 0.03) was detected for MPO due to its upregulation at -15 d in the AAC
cows (Table 2, Fig. 1). The expression of the superoxide dismutase enzymes SOD1 (P = 0.14), SOD2 (P = 0.27) and
SOD3 (P = 0.15) was not affected by treatment. However,
the expression of SOD1 increased from -15 to +10 d regardless of treatment (Table 2, Fig. 1). The oxidant scavenger proteins S100A8 (P = 0.78) and NOX1 (P = 0.94) as
well as the transcription factor NFE2L2 (P = 0.54) were not
affected by treatments (Table 2, Fig. 1). However, S100A8
was upregulated (P = 0.08) from -15 to +10 d in both
treatments.
Glucose metabolism
An interaction T × T (P < 0.01) was detected for the glucose transporter SLC2A1 due to the marked decrease in
expression between -15 and +10 d. Whereas, the mRNA
expression of LDHA (P = 0.06) tended to be lower in the
AAC compared with INO cows (Table 2, Fig. 2).
Endometrium
Inflammation response
A tendency for a T × T interaction was detected for
TLR2 (P = 0.08), TLR4 (P = 0.08), TNF (P = 0.10), and
NFKB1 (P = 0.06) due to greater mRNA expression in
AAC cows at +14 d, whereas lower expression was observed at +30 d (Table 3, Fig. 3). Furthermore, a T × T
was observed for IL6 (P = 0.03), IL1B (P < 0.01), IL8
(P < 0.01), and IL10 (P < 0.01) because all these genes had
greater mRNA expression in the AAC cows at +14 d but
no treatment effect was detected at +30 d. In addition,
AAC treatment increased STAT3 (P = 0.05) and tended to
increase MYD88 (P = 0.06) mRNA expression (Table 3).
The expression of MYD88 decreased (P = 0.05) regardless
of treatment from +14 to +30 d postpartum.
Oxidative stress
Eicosanoids
Among the genes related with arachidonic acid, PLA2G4A
(P = 0.06) and ALOX5AP (P = 0.09) tended to have greater
expression in cows fed AAC (Table 2, Fig. 1). In contrast,
the mRNA expression of LTA4H (P = 0.76) and PTGS2
(P = 0.53) were not affected by treatments (Table 2,
Fig. 1). A marked decrease (P < 0.01) in expression of
LTA4H was observed between -15 and +10 d regardless
of treatment, whereas PTGS2 expression gradually decreased (P = 0.04) between -15 and +30 d regardless of
treatment (Fig. 1).
Transcription factors
PPARA had lower overall mRNA expression (P = 0.04)
in AAC cows, whereas PPARG tended to have greater
mRNA expression compared with INO (P = 0.09; Table 2
and Fig. 2). The mRNA expression of PPARD (P = 0.71)
and RXRA (P = 0.22) were not affected by treatments
(Table 2). However, expression of RXRA increased (P =
0.05) from -15 to +10 d and then decreased to prepartum values in both treatments (Fig. 2).
No T × T was observed (P > 0.10) for genes related with
oxidative stress (Table 3). However, mRNA expression of
SOD1 (P = 0.09) and NEF2L2 (P = 0.06) tended to be
greater in AAC compared with INO cows (Table 3). In
addition, expression of SOD2 decreased (P < 0.01) regardless of treatment from +14 to +30 d postpartum.
Eicosanoids
A T × T (P = 0.04) was observed for PTGS2 because its expression became greater over time with INO while it decreased with AAC (Table 3, Fig. 3). A tendency (P = 0.10)
for a greater overall PTGES mRNA expression was observed for AAC cows (Table 3). In addition, expression of
PTGES decreased (P < 0.01) from +14 to +30 d postpartum regardless of treatment (Table 3).
Transcription factors
No T × T or overall treatment effect (P > 0.10) was observed for PPARA, PPARD, PPARG and RXRA. However,
expression of PPARA and PPARG increased and PPARD
decreased from +14 to +30 d postpartum (Table 3).
Receptors
Antimicrobial peptides
The expression of the receptors TLN1 (P = 0.01) and
ADORA1 (P = 0.01) was greater in the cows receiving
MUC1 mRNA expression was upregulated (P = 0.04)
overall in the AAC compared with INO cows (Table 3).
Batistel et al. Journal of Animal Science and Biotechnology (2017) 8:33
Page 6 of 12
Inflammation
Oxidative stress
2.8
2.0
INO
AAC
1.5
MPO
DDX58
2.2
1.6
1.0
0.5
0.4
0.0
1.3
3.0
1.1
2.5
S100A8
NFKB1
1.0
1.0
2.0
0.9
1.5
0.7
1.5
1.0
1.6
1.2
SOD1
ZBP1
1.4
0.9
1.2
0.6
1.0
0.3
0.8
1.5
1.6
1.4
PLA2G4A
1.9
1.3
1.0
1.2
1.1
0.7
0.9
1.3
2.4
1.0
1.8
PTGS2
LTA4H
ALOX5AP
Eicosanoids
0.8
0.5
1.2
0.6
0.3
0.0
-15
10
30
Day relative to parturition
-15
10
30
Day relative to parturition
Fig. 1 mRNA expression (fold-change relative to -30 d prepartum) of genes associated with inflammation (DDX58, NFKB1 and ZBP1), oxidative
stress (MPO, S100A8 and SOD1) and eicosanoids (ALOX5AP, LTA4H, PLA2G4A and PTGS2) in polymorphonuclear leukocytes (PMNL) of cows supplemented
with inorganic (INO; n = 11) or complexed (AAC; n = 9) trace minerals during the pre- and postpartal period
Discussion
Neutrophil function around calving
Neutrophil function and bactericidal efficiency is compromised during the transition period [1]. A lower capacity for trafficking, phagocytosis, and pathogen killing
during this period is partly associated with changes in
hormones and metabolites and with immune or stresslike conditions [19]. Some studies also have reported
that neutrophils have impaired generation of ROM
during the transition period [20, 21], a feature that
may contribute to greater susceptibility to disorders in
early lactation. The general hypothesis of this study
was that feeding trace minerals through more bioavailable forms would improve immune health, enhance
PMNL activity, and help overcome the challenges of
the transition period [10]. In the companion paper [10]
we reported production and biomarker data indicating
that better DMI and milk production in cows fed AAC
was partly due to better liver function and PMNL
phagocytosis.
Batistel et al. Journal of Animal Science and Biotechnology (2017) 8:33
Page 7 of 12
Receptors
Transcription factors
2.0
2.4
INO
AAC
ADORA1
2.0
1.2
1.2
0.4
0.8
2.8
1.1
2.2
1.0
1.6
1.0
0.8
0.7
0.4
0.5
1.5
1.5
1.3
1.3
PANX1
RXRA
1.6
0.8
ITGAM
PPARG
PPARA
1.6
1.1
0.9
1.0
0.8
0.7
0.5
1.4
1.5
1.2
TLN1
1.9
1.2
1.0
0.9
0.8
0.5
0.6
1.2
1.2
1.0
1.0
VCL
SLC2A1
LDHA
Glucose metabolism
0.7
0.5
0.8
0.6
0.2
0.4
-15
10
30
Day relative to parturition
-15
10
30
Day relative to parturition
Fig. 2 mRNA expression (fold-change relative to -30 d prepartum) of genes associated with transcription factors (PPARA, PPARG and RXRA), receptors
(ADORA1, ITGAM, PANX1, TLN1 and VCL) and glucose metabolism (LDHA and SLC1A1) in polymorphonuclear leukocytes (PMNL) of cows supplemented
with inorganic (INO; n = 11) or complexed (AAC; n = 9) trace minerals during the pre- and postpartal period
Inflammation response
The activation of innate immune responses are regulated
by several DNA sensors including toll-like receptors,
e.g., TLR2, TLR4, DDX58 and ZBP1 [22]. Subsequent to
pathogen detection, these receptors activate signaling
pathways (e.g., STAT3 and NF-κB), which trigger the
synthesis of pro-inflammatory cytokines and chemokines
[23]. Despite the fact that cows in the AAC treatment
had greater expression of DDX58 and lower expression
of ZBP1, those responses did not seem to activate the
pro-inflammatory pathways as indicated by the lack of
change in expression of the cytokines TNF and IL1B.
Oxidative stress
Myeloperoxidase (MPO) is the main peroxidase enzyme
released upon neutrophil activation, and catalyzes the
formation of hypochlorous acid, a potent oxidant that
displays bactericidal activity [24]. Furthermore, MPO
Batistel et al. Journal of Animal Science and Biotechnology (2017) 8:33
Gene
SEMa P value1
Treatments
INO
AAC
+14 +30
+14 +30
Treatment Time
Eicosanoids
6
4
PTGS2
Table 3 Effects of supplementing cows with inorganic (INO; n =
11) or complexed (AAC; n = 9) trace minerals during the peripartal
period on mRNA expression (log-2 scale) of genes related with
inflammation response, oxidative stress, eicosanoids, transcription
factors and antimicrobial peptides in endometrium tissue at +14
and +30 d after parturition
Page 8 of 12
2
T × Tb
0
Inflammation
IL10
4.13 3.83
0.19
0.04
<0.01 <0.01
IL1B
0.65 -0.37 3.51 -0.98 0.84
5.13 3.72
0.26
<0.01 <0.01
IL6
2.12 2.91
0.23
0.24
0.15
IL8
1.26 -0.01 4.51 -1.52 0.84
0.37
<0.01 <0.01
MYD88
4.98 4.86
5.46 4.93
0.15
0.06
0.05
0.20
NFKB1
5.06 5.36
5.41 5.06
0.16
0.87
0.90
0.06
SAA3
3.10 2.77
4.88 2.06
1.00
0.46
0.15
0.25
STAT3
5.05 5.22
5.69 5.16
0.19
0.05
0.40
0.11
Inflammation
Inflammation
6
0.03
5.05 4.58
5.87 4.39
0.28
0.19
<0.01 0.10
TLR4
5.24 4.49
5.69 3.34
0.18
0.40
<0.01 0.08
TNF
4.21 4.60
5.67 4.32
0.36
0.11
0.12
0.01
Oxidative stress
IL10
IL1B
4
2
2
0
-2
0
6
8
6
4
TLR2
TLR2
4
IL6
2.88 2.70
6
2
4
NFE2L2
4.85 5.11
5.20 5.26
0.13
0.06
0.22
0.43
NOS3
4.94 5.16
5.11 5.07
0.36
0.91
0.76
0.66
NRROS
5.18 4.62
5.20 4.87
0.32
0.68
0.13
0.70
SOD1
5.19 5.04
5.26 5.28
0.09
0.09
0.39
0.30
6
SOD2
4.83 4.13
5.57 3.83
0.32
0.44
<0.01 0.12
4
SOD3
4.87 5.27
5.08 5.16
0.26
0.85
0.28
0.45
2
4.89 4.83
5.08 5.14
0.41
0.51
0.99
0.88
ALOX5AP 4.86 4.92
5.33 4.95
0.38
0.51
0.60
0.48
LTA4H
5.07 5.30
5.24 5.23
0.10
0.51
0.30
0.24
-4
0
LTC4S
3.91 3.87
4.35 3.98
0.42
0.45
0.62
0.70
8
8
PLA2G4A 4.92 5.39
5.44 4.96
0.29
0.86
0.99
0.13
6
PTGDS
5.26 4.94
5.05 4.87
0.29
0.63
0.34
0.77
6
PTGES
4.50 3.68
5.53 3.92
0.34
0.10
<0.01 0.15
PTGS2
3.17 4.32
4.94 2.85
0.77
0.83
0.52
2
0
0
8
0.04
PPARA
5.09 5.48
5.20 5.68
0.21
0.48
0.02
0.79
PPARD
5.47 5.13
5.46 5.07
0.15
0.81
0.01
0.89
PPARG
4.72 5.17
4.64 6.03
0.31
0.21
<0.01 0.11
RXRA
5.28 5.24
5.24 5.09
0.13
0.49
0.38
0.60
4.29 4.05
0.46
0.04
0.49
0.25
Antimicrobial peptides
MUC1
2.84 3.78
P values represents the probability of statistical significance for the fixed
effects (treatment, time, treatment × time). Statistical differences were
declared significant at P ≤ 0.05 and tendencies at P ≤ 0.10
a
Largest standard error of the mean is shown
b
Interaction of treatment × time
4
2
-2
4
2
Transcription regulation
1
0
TNF
ALOX5
NFKB1
Eicosanoid synthesis
TLR4
IL8
6
4
2
0
0
14
30
14
30
Fig. 3 mRNA expression (log 2-scale) of genes associated with immunerelated receptors (TLR2 and TLR4), pro-inflammatory response (NFKB1,
TNF, IL6, IL1B and IL8), anti-inflammatory response (IL10) and eicosanoids
(PTGS2) in endometrium of cows supplemented with inorganic
(INO; n = 11) or complexed (AAC; n = 9) trace minerals during
the pre- and postpartal period
Batistel et al. Journal of Animal Science and Biotechnology (2017) 8:33
Page 9 of 12
has traditional cytokine-like function and acts as an
autocrine modulator of neutrophil activation [25]. In
steers, a Cu-deficient diet impaired neutrophil killing
capacity without altering phagocytosis [26]. Similarly,
Cu-depleted calves exhibited impaired phagocytic killing
activity, which was restored by Cu supplementation [27].
Therefore, the greater mRNA expression of MPO indicated that AAC cows were more likely to have greater
PMNL activation, hence, superior capacity to kill invading pathogens.
When ROM production elicits a metabolic imbalance
in cells, the release of endogenous neutralizing agents
helps to minimize their potential deleterious effects. The
protein S100A8 comprises ~20% of the PMN cytoplasm
[28], and exerts an important protective mechanism during inflammation because it scavenges intracellular
ROM produced by activated PMN and attenuates nitric
oxide production [29]. Similarly, the cytoplasmic enzyme
SOD1 transforms the harmful superoxide radicals to
molecular oxygen and hydrogen peroxide [30]. Therefore, the tendency for upregulation of SOD1 and S100A8
at +10 d regardless of treatment could be taken as an indication of PMNL attempting to neutralize the greater
oxidative stress experienced after parturition [31].
degradation of leukotriene B4 [37]. It is noteworthy that
the expression of ALOX5AP (leukotriene synthesis) and
PPARA followed opposite patterns of expression with the
AAC treatment indicating that the inflammatory response
in those cows likely was of a greater magnitude but of
brief duration. The absence of change in the expression of
the pro-inflammatory cytokine IL1B may be due to the
upregulation of PPARG in the AAC treatment. Prior research in non-ruminant cells indicated an inhibitory effect
of PPARγ on cytokine production [34].
Eicosanoid metabolism
Neutrophil stimulation produces oxygen-derived reactive species, lysosomal enzymes, nitric oxide as well as
pro-inflammatory and anti-inflammatory mediators
which include bioactive lipids such as the eicosanoids
(e.g., prostaglandins and leukotrienes) [32]. The tendency for upregulation of PLA2G4A in AAC cows
could have resulted in an increase in the hydrolysis of
cell membrane phospholipids to release arachidonic
acid, which subsequently could be used for leukotriene (via ALOX5AP) synthesis. Leukotrienes, such as
leukotriene B4, are essential components of the inflammatory response because they act as chemoattractants for mature neutrophils, and promote neutrophil
activation [33]. Furthermore, leukotriene B4 enhances
cytokine production and the presence of these fatty
acids seems to determine the duration and magnitude
of the inflammatory response [34]. In vitro, Zn, Cu,
and Ni enhanced PMN motility by chemotactic activation indicating that the inflammatory response can
be partly modulated through the availability of those
metals [35].
Transcription regulation
In non-ruminants, the family of transcription factors
termed peroxisome proliferator-activated receptors
(PPAR) is involved in the control of inflammation [34]. Eicosanoids are PPARα activators [36] that can inhibit arachidonic acid-induced inflammation in part by enhancing
Receptors
Neutrophil recruitment and migration to inflamed tissues
are critical for proper immune function. Cytoskeletal proteins, such as talin-1 (TLN1), facilitate the transition from
neutrophil rolling to arrest [38]. Furthermore, modulation
of neutrophil function by adenosine (ADORA1) promotes
neutrophil chemotaxis and phagocytosis [39]. Therefore,
the upregulation of TLN1 and ADORA1 in AAC cows indicated that PMNL were better equipped to be deployed into
the inflamed sites. Despite these unique effects of AAC, the
overall downregulation of PANX1 and VCL from -15 to
+10 d regardless of treatment seemed to indicate a degree
of impairment in the recruitment of PMNL and their ability
to adhere to endothelium. Some evidence indicates that
PANX1 channels are activated by ATP [40], which may explain the gradual upregulation of ADORA1 over time, i.e., a
counter regulatory mechanism to help regulate PMNL activity. In addition, the downregulation of VCL could partly
be explained by gradual degradation of PMNL plasma
membrane phosphatidylinositol 4,5-bisphosphate, which is
essential for activation of VCL [41]. Whether such effect is
directly related to catabolic enzymes (e.g., phospholipases)
or greater turnover of PMNL is unknown.
Glucose metabolism
At least in non-ruminants, neutrophils rely on glycolysis
as the main source of energy; however, the extra energy
required for phagocytosis is usually derived from metabolism of lactate [42]. Both SCL2A1 and LDHA are important regulators of energy metabolism in neutrophils.
The first facilitates the transport of glucose across the
plasma membrane, whereas the second is involved in the
interconversion of pyruvate to lactate after glycolysis
[43]. The parallel downregulation of SLC2A1 and LDHA
regardless of treatment to a nadir at +10 d postpartum
was most likely a result of the shortfall in circulating
glucose commonly observed after parturition [10]. The
numerically-greater expression of SLC2A1 and LDHA in
INO cows during the study could indicate that these
cows were more immuno-compromised because a previous study detected marked upregulation of LDHA in
PMNL after a mastitis challenge [14]. If such an effect
existed it could help to partly explain the lower
Batistel et al. Journal of Animal Science and Biotechnology (2017) 8:33
Page 10 of 12
phagocytic activity in whole blood that was measured on
+30 d in INO cows [10]. Because cows in INO had
greater plasma concentrations of ketones, the upregulation of LDHA in these cows could have been a
mechanism induced by ketone body (e.g., hydroxybutyrate) metabolism to decrease glucose oxidation by
the PMNL [44].
oxidative stress within the endometrium and potentially help alleviate an overt inflammatory response. It
is noteworthy that upregulation of NFE2L2 also occurred in the PMNL and hoof corium (unpublished
results) in the cows fed AAC, which strongly indicates a consistent effect of trace minerals on cellular
stress through this transcription regulator.
Endometrium
Bacterial contamination and consequent inflammatory
response of the uterine tissue after parturition are common and are associated with lower conception rates,
longer interval periods from calving to first service or
conception, and more animals culled for failure to conceive [45]. Considering that trace minerals play important roles in the health and immunity of peripartal dairy
cows [46] and complexed trace mineral supplementation
in partial substitution of sulfate sources elicited an improvement in immune function [10, 47], it was important to ascertain if complexed trace minerals also elicited
a local response in the endometrium.
Eicosanoid metabolism
Among several biological functions, prostaglandins play
a central role in the generation of an inflammatory response. They have pro-inflammatory properties and are
responsible for typical signs of inflammation including
redness, swelling and pain [50]. The synthesis of prostaglandins is partly dependent on Zn, hence, this trace
mineral could play an indirect role in regulating enzymes involved in the arachidonic acid cascade that result in production of prostaglandins [51, 52]. Therefore,
the upregulation of PTGS2 and PTGES in cows fed
AAC indicated that supplemental complexed Zn was
more bioavailable for prostaglandin synthesis.
Inflammation response
Although the inflammatory response is a natural defense
mechanism that could be initiated by tissue injury [22],
it can be beneficial or deleterious. After calving, the inflammatory and immune response in the endometrium
attempts to eliminate any pathogenic bacterial contamination as well as initiate tissue repair as part of the involution process [45]. However, prolonged inflammation
and cytokine production within the reproductive tract
impair immune status and reproductive performance
[45]. Therefore, the upregulation of genes related with
the pro-inflammatory cascade (TLR2, TLR4, NFKB1,
TNF, IL6 and IL1B) in response to AAC at +14 d compared with +30 d indicated that pathogen elimination
and tissue remodeling processes occurred earlier than in
INO cows. In addition, the concentrations of blood biomarkers of inflammation [47] in these cows indicated a
lower systemic inflammation status in AAC than INO
cows.
Oxidative stress response
Essential trace minerals such as Zn and Cu play a
central role in metabolism and have the potential to
reduce oxidative stress through several mechanisms.
Evidence from human studies suggest that Zn is essential for expression and function of the transcription factor NFE2L2 [48]. This transcription factor
helps control oxidative damage through its control of
some antioxidant defense systems such as SOD1 activity [49] which requires Zn and Cu as co-factors.
Therefore, the overall greater expression of NFE2L2
and SOD1 indicated that feeding AAC reduced
Antimicrobial peptides
The upregulation of MUC1, a transmembrane glycoprotein abundantly expressed at the surface of the uterine epithelial tissue, in AAC cows indicated a greater
ability to eliminate invading pathogenic bacteria [53].
This idea is supported by previous work demonstrating
that MUC1-null mice were susceptible to chronic infections and inflammation, and had a markedly reduced
fertility [54].
Conclusions
Taken together, our findings reveal that supplementation
with Zn, Mn, and Cu from AA complexes and Co from
Co glucoheptonate during the transition period improved PMNL function and likely confer these cells a
greater capacity to control invading pathogens. The robust inflammatory response coupled with the antioxidant response discerned from the transcriptome data
in the uterine samples of cows fed complexed trace minerals likely allowed for a faster uterine recovery. These
data indicate that the benefits of trace minerals from AA
complexes extend beyond an improvement of liver function and productive performance [10]. Although our
findings suggest that peripheral and uterine immune
function was improved in cows supplemented with more
bioavailable forms of trace minerals, further research to
evaluate the clinical impact of that supplementation is
warranted. Such research also will help to better define
complexed trace mineral requirements beyond productive purposes.
Batistel et al. Journal of Animal Science and Biotechnology (2017) 8:33
Additional files
Additional file 1: PMNL Isolation: Blood (120 mL), collected in ACD
solution A vacutainer tubes, was mixed well by inversion and placed on
ice until PMN isolation (within ~30 min). Tubes were combined into
three 50-mL conical tubes (Fisher Scientific, Pittsburgh, PA) and
centrifuged at 918 × g for 30 min at 4 °C. The plasma, buffy coat, and
approximately one-third of the red blood cells (RBC) were removed and
discarded. Cells were lysed with 25 mL of deionized water at 4 °C,
homogenized gently by inversion, and then 5 mL of 5 × PBS (pH 7.4) at
4 °C was added, in order to restore an iso-osmotic environment. The cell
suspension was centrifuged at 330 × g for 10 min at 4 °C and the
supernatants were decanted. Ten milliliters of 1 × PBS at 4 °C was added
in each tube, homogenized until there was nothing attached to the
bottom of the tube, and then the three tubes were combined in one.
The cell suspension was centrifuged at 663 × g for 5 min at 4 °C and the
supernatants were discarded. The remaining RBC were lysed with 8 mL
of deionized water at 4 °C, homogenized gently by inversion and 2 mL
of 5 × PBS at 4 °C was added. The samples were centrifuged at 663 × g
for 5 min at 4 °C and the supernatant was discarded. Two subsequent
washings using 10 mL of 1 × PBS at 4 °C were performed, centrifuged at
663 × g for 5 min at 4 °C and supernatant discarded. Prior to the last
centrifugation, 100 μL of the cell suspension were aliquoted for further
PMN concentration and cell viability analysis. (DOC 43 kb)
Additional file 2: RNA extraction: Approximately 40 mg of frozen tissue
was weighed and immediately placed in ice-cold 1 mL Qiazol reagent
(Qiagen 75842; Qiagen Inc., Valencia, CA) for homogenization. After
homogenization, the samples were centrifuged for 10 min at 12,000 × g
at 4 °C to remove the insoluble material. The supernatant was transferred
to a collection tube and incubated for 5 min on ice. Chloroform (200 µL)
was added to each tube and the sample incubated at room temperature
for 3 min. Subsequently, samples were centrifuged for 15 min at 12,000 ×
g at 4 °C, and the upper phase was transferred to a new collection tube
without disturbing the mid and lower phases. A second wash was
performed with 100% ethanol; 750 µL was added and transferred to a
miRNeasy Mini Kit columns (Cat. No: 217004, Qiagen). Genomic DNA was
removed on column from RNA samples with RNase-free DNase I, using
the recommended protocol provided with the miRNeasy Mini Kit. RNA
concentration was measured with a NanoDrop ND-1000 spectrophotometer
(Thermo Fischer Scientific; Wilmington, DE), while the RNA quality was
assessed using the Agilent 2100 Bioanalyzer system (Agilent Technologies,
Santa Clara, CA). Samples of RNA used for analysis had an RNA integrity
number ≥7.0. (DOC 44 kb)
Additional file 3: Function of the genes measured in the PMNL.
(DOC 67 kb)
Page 11 of 12
Corporation provided support to Juan J. Loor and Michael T. Socha. Zinpro
Corporation had a role in the study design and provided financial support to
cover costs of animal use, data collection, and sample analyses. The specific
roles of the authors are articulated in the ‘author’s contributions’ section.
Funding
Not applicable.
Availability of data and materials
The datasets during and/or analyzed during the current study are available
from the corresponding author on reasonable request.
Authors’ contributions
FB, JSO, MRT, CL, and JC performed analyses and analyzed data. JJL, JSO, and
MTS conceived the animal experiments. FB wrote the manuscript. All authors
approved the final version of the manuscript.
Authors’ information
F. Batistel is PhD candidate, University of Illinois, Urbana, Illinois, 61801, USA.
J. S. Osorio is Assistant Professor in the Department of Dairy Science, South
Dakota State University, Brookings, South Dakota, USA. M. R. Tariq is Assistant
Professor in the Department of Food Science and Technology, University
College of Agriculture & Environmental Sciences, The Islamia University of
Bahawalpur, Bahawalpur, Punjab, Pakistan. C. Li, PhD, College of Animal
Science and Technology, Key Laboratory of Animal Genetics and Breeding of
Ministry of Agriculture, National Engineering Laboratory for Animal Breeding,
China Agricultural University, Beijing 100193, China. J. Caputo, PhD, University
of Illinois, Urbana, Illinois, 61801, USA. M. T. Socha, PhD, is Regional RNS
Manager-North America, Research and Nutritional Services, Zinpro Corporation, 10400 Viking Dr., Suite 240, Eden Prairie, Minnesota 55344, USA. J. J.
Loor is Associate Professor in the Department of Animal Sciences, University
of Illinois, Urbana, Illinois, 61801, USA.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
Not applicable.
Ethics approval
All procedures for this study (protocol no. 12097) were approved by the
Institutional Animal Care and Use Committee of the University of Illinois.
Additional file 6: Sequencing results of PCR products from primers of
genes used for this experiment. (DOC 62 kb)
Author details
1
Department of Animal Sciences and Division of Nutritional Sciences,
University of Illinois, 1207 West Gregory Drive, Urbana, IL 61801, USA.
2
Department of Dairy Science, South Dakota State University, Brookings, SD,
USA. 3Department of Food Science and Technology, University College of
Agriculture & Environmental Sciences, The Islamia University of Bahawalpur,
Bahawalpur, Punjab, Pakistan. 4College of Animal Science and Technology,
Key Laboratory of Animal Genetics and Breeding of Ministry of Agriculture,
National Engineering Laboratory for Animal Breeding, China Agricultural
University, Beijing 100193, China. 5Zinpro Corporation, Eden Prairie, MN, USA.
Additional file 7: qPCR performance among the genes measured in
PMNL. (DOC 77 kb)
Received: 24 December 2016 Accepted: 23 March 2017
Additional file 4: Function of the genes measured in the endometrium.
(DOC 61 kb)
Additional file 5: Features of used primers for qPCR analysis. Hybridization
position, sequence, and amplicon size of primers for Bos taurus used to
analyze gene expression. (DOC 107 kb)
Additional file 8: qPCR performance among the genes measured in the
endometrium tissue. (DOC 71 kb)
Abbreviations
AAC: Amino acid-complexed Zn, Mn, Cu, and Co; ACD: Citric acid and
dextrose; DIM: Days in milk; DMI: Dry matter intake; IACUC: Institutional
Animal Care and Use Committee; INO: Inorganic trace mineral mix;
PMNL: Polymorphonuclear leukocyte; PPAR: Peroxisome proliferator-activated
receptors; ROM: Reactive oxygen metabolites
Acknowledgments
Fernanda Batistel (FB) was supported by Coordenação de Aperfeiçoamento
de Pessoal de Nível Superior (CAPES). Juan Loor (JL) was supported by
National Institute of Food and Agriculture (Grant: ILLU-538-914). Zinpro
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