J. Dairy Sci.
104
https://doi.org/10.3168/jds.2021-20284
© 2021 American Dairy Science Association®. Published by Elsevier Inc. and Fass Inc. All rights reserved.
Effect of source and amount of vitamin D on function
and mRNA expression in immune cells in dairy cows
A. Vieira-Neto,1,2* M. B. Poindexter,1 M. Nehme Marinho,1 R. Zimpel,1,2 A. Husnain,1 A. C. M. Silva,1 J. G. Prim,1
C. D. Nelson,1 and J. E. P. Santos1,2†
1
Department of Animal Sciences, University of Florida, Gainesville 32611
2
DH Barron Reproductive and Perinatal Biology Research Program, University of Florida, Gainesville 32611
ABSTRACT
Objectives were to determine the effect of supple-
menting 2 sources of vitamin D, cholecalciferol (CH)
or calcidiol (CA), at 1 (1mg) or 3 mg/d (3mg) pre-
partum on concentrations of vitamin D metabolites
in plasma, measures of innate immune function, and
leukocyte mRNA expression. Parous Holstein cows (n
= 99) were assigned to a daily treatment administered
as top-dress containing either 1 or 3 mg of CH (CH1 or
CH3) or of CA (CA1 or CA3) from 250 d of gestation
until calving. Plasma concentrations of vitamin D, im-
mune cell population in blood, cell adhesion markers,
and granulocyte phagocytosis and oxidative burst were
evaluated pre- and postpartum. The mRNA expression
in leukocytes was determined at 270 d of gestation
and 3 d postpartum for genes involved in cell migra-
tion, pathogen recognition receptors, cell signaling,
cytokines, antimicrobial mechanisms, oxidative burst,
and Ca and vitamin D metabolism. Concentrations of
vitamin D3 increased in cows fed CH, whereas those of
25-hydroxyvitamin D3 increased in cows fed CA. Per-
centage of granulocytes from total leukocytes differed
with amount of vitamin D pre- (1mg = 24.5 vs. 3mg =
37.9%) and postpartum (1mg = 22.0 vs. 3mg = 31.0%),
thus shifting mononuclear cells in the opposite direction
pre- (1mg = 75.5 vs. 3mg = 62.1%) and postpartum
(1mg = 78.0 vs. 3mg = 69.0%). Granulocytes display-
ing phagocytosis (1mg = 69.0 vs. 3mg = 62.9%) and in-
tensity of phagocytosis prepartum (1mg = 7.46 vs. 3mg
= 7.28) tended to be less in cows fed 3mg compared
with 1mg. During prepartum, CA increased mRNA ex-
pression of genes related to cell adhesion and migration
(CD44, ICAM1, ITGAL, ITGB1, LGALS8, SELL),
pathogen recognition receptor (NOD2, TLR2, TLR6),
Received February 10, 2021.
Accepted May 31, 2021.
*Present address: Department of Animal Sciences and Industry,
Kansas State University, Manhattan 66506.
†Corresponding author: jepsantos@​ufl​.edu
cell signaling (FOS, JUN, NFKB2), cytokine signal-
ing (IL1B, IL1R1, IL1RN), antimicrobial mechanisms
(CTSB, LYZ), and Ca metabolism (ATP2B1, STIM1,
TRPV5) compared with CH. Similarly, postpartum,
CA increased mRNA expression of genes related to cell
adhesion and migration (CXCR2, SELL, TLN1), cell
signaling (AKT2), cytokines (CCL2, IL1R1, ILRN),
antimicrobial mechanisms (DEFB3), oxidative burst
(RAC2), and calcium metabolism (CALM3) compared
with CH. Feeding additional vitamin D in the last 3 wk
of gestation changed the profile of blood leukocytes and
attenuated granulocyte phagocytosis during the tran-
sition period, whereas supplementing CA prepartum
increased mRNA expression of genes involved in im-
mune cell function, including genes related to pathogen
recognition and antimicrobial effects of leukocytes.
Key words: calcidiol, dairy cow, immunity, vitamin D
INTRODUCTION
Micronutrients such as fat-soluble vitamins play an
important role regulating the immune response, and
vitamin D affects mineral metabolism and innate
immune defenses (Nelson et al., 2010; Rodney et al.,
2018). Vitamin D3, also known as cholecalciferol (CH),
can be endogenously synthesized in the skin when the
latter is exposed to sunlight, or it can be acquired
from the diet. In dairy cattle, supplementation with
CH is a common practice (Nelson et al., 2016) and,
once absorbed, CH is hydroxylated by 25-hydroxylase
in the liver to 25-hydroxyvitamin D3, also known as
calcidiol (CA), the more stable metabolite of vitamin
D. The most abundant circulating form of vitamin D
is 25-hydroxyvitamin D3 and therefore is used as an
indicator of the vitamin status of the animal (Nelson
et al., 2016). In the kidney, 25-hydroxyvitamin D3 is
further hydroxylated by 1α-hydroxylase to form 1α,25-
dihydroxyvitamin D3, also known as calcitriol, the
steroid hormone that is the most active metabolite of
vitamin D. This final conversion is a tightly regulated
process (Fraser and Kodicek, 1973). Inactivation of
 Vieira-Neto et al.: SOURCE AND AMOUNT OF VITAMIN D AND IMMUNE CELLS
25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 is
carried out by 24-hydroxylase, producing 24,25-dihy-
droxyvitamin D3.
Plasma concentrations of vitamin D metabolites are
responsive to the amount of dietary supplementation,
although increasing serum 25-hydroxyvitamin D3 can
be more easily achieved by supplementing CA than CH
(Rodney et al., 2018; Poindexter et al., 2020). Even at
almost 4 times the current NRC (2001) recommenda-
tions, supplementing CH did not increase concentra-
tions of 25-hydroxyvitamin D3 in plasma above 100 ng/
mL, suggesting a regulatory control on the conversion
of vitamin D3 into 25-hydroxyvitamin D3 by the he-
patic 25-hydroxylase (Rodney et al., 2018; Poindexter
et al., 2020). Therefore, it is possible that the effects
of vitamin D supplementation may be enhanced by
supplementing CA compared with CH. It is clear that
adequate vitamin D status, based on concentrations of
25-hydroxyvitamin D3 in plasma (Nelson et al., 2016),
can be more easily achieved by supplementing CA than
CH.
Immune cells and other tissues express 1α-hydroxylase
and can regulate local synthesis of calcitriol. Nelson et
al. (2010) demonstrated that pathogen-associated mol-
ecules such as LPS and peptidoglycan can stimulate
local expression of 1α-hydroxylase in peripheral blood
monocytes in dairy cows. In addition, intramammary
endotoxin challenge in dairy cows resulted in activa-
tion of the vitamin D signaling pathway in mammary
gland macrophages and neutrophils (Merriman et al.,
2018). In fact, in vitro data demonstrated that CA up-
regulates mRNA expression of important antimicrobial
peptides, β-defensins, in monocytes (Merriman et al.,
2015). Thus, it is possible that supplementation with
CA could increase substrate availability for local syn-
thesis of calcitriol in immune cells, which might benefit
immune response. In support of that, calcitriol has
been shown to increase β-defensins and inducible nitric
oxide synthase mRNA in milk somatic cells (Merriman
et al., 2017). Interestingly, supplementation of dietary
CA prepartum resulted in increased concentrations of
CA in plasma in dairy cows pre- and postpartum and
reduced incidence of retained placenta and metritis
(Martinez et al., 2018). It is possible that CA influ-
ences leukocyte mRNA expression for key regulatory
proteins involved in vitamin D metabolism and immune
cell functions important for pathogen clearance.
The hypothesis was that supplementing CA would
increase concentrations of 25-hydroxyvitamin D3 in
plasma and enhance measures of innate immune cell
function by altering mRNA expression and intensity
of expression of surface markers linked with innate
immune response in peripheral blood leukocytes, com-
pared with CH. It was anticipated that increasing the
Journal of Dairy Science Vol. 104 No. 10, 2021
amount of vitamin D would result in increased effects
when the source was CA rather than CH. Therefore,
the objective of this experiment was to determine the
effects of 2 sources of vitamin D3, CH or CA, supple-
mented at 2 amounts, 1 or 3 mg/d, during the last 3
wk of gestation on granulocyte function and peripheral
blood leukocyte mRNA expression in dairy cows.
MATERIALS AND METHODS
The experiment was conducted at the University of
Florida (Gainesville) Dairy Research Unit. Procedures
for animal handling and care were approved by the
University of Florida Institutional Animal Care and
Use Committee under the protocol number 20171002.
These data are part of an experiment evaluating the
effects of vitamin D on performance and health that
included 170 transition Holstein cows (Poindexter et
al., 2019).
Experimental Design and Sample Size Calculation
The experiment was a randomized complete block
design with a 2 × 2 factorial arrangement of treatments
and cow as the experimental unit. A 2-sided sample
size was calculated for 4 treatments using α = 0.05 and
β = 0.20 to allow detection of a 1-unit difference in
delta cycle threshold (dCt) in mRNA expression, as-
suming that the standard deviation (SD) for dCt would
be a maximum of 1.0 unit. The sample size calculated
was 23 experimental units per treatment. Additional
cows were enrolled to account for potential attrition at
calving. Because of the arrangement of treatments as
a 2 × 2 factorial, the interaction term would result in
more than 95% power to detect a difference in mRNA
expression, assuming a 1-unit difference in dCt and an
SD for dCt of 1.0 unit.
Cows, Housing, Treatments, and Diets
A total of 99 parous Holstein cows were moved to
the experimental facilities at 242 d of gestation, and
dietary treatments started at exactly 250 d of gestation
of each cow and ended at calving. Prepartum cows were
housed in a freestall barn equipped with individual feed-
ing gates (Calan Broadbent feeding system, American
Calan Inc.) that were assigned randomly to each cow.
Individual cow DMI was measured daily prepartum.
Postpartum, cows were housed in a single group in a
freestall barn for the first 42 d in lactation.
Every 2 wk, a cohort of 8 to 12 parous cows at 240
d of gestation were blocked based on parity group (1
vs. >1) and then by 305-d milk yield in the recently
completed lactation and, within each block, assigned
 Vieira-Neto et al.: SOURCE AND AMOUNT OF VITAMIN D AND IMMUNE CELLS

randomly to 1 of 4 treatments. Treatments included 2 Table 1. Ingredient composition and nutrient content of the prepartum
diet1
sources of vitamin D, CH (Rovimix D3, 12.5 g of CH
per kg; Division of Animal Nutrition and Health, DSM Item Value
Nutritional Products LLC) or CA (Hy-D, 12.5 g of CA Ingredient, % of DM
per kg; DSM Nutritional Products LLC), supplemented   Corn silage 50.0
at 2 amounts, 1 or 3 mg/d arranged as factorial, result-   Bermuda grass hay 23.1
ing in 4 treatments: CH at 1 mg/d (CH1, n = 21), CH   Soybean meal, solvent extract 7.7
  Citrus pulp, dry 9.2
at 3 mg/d (CH3, n = 27), CA at 1 mg/d (CA1, n =   Acidogenic product2 5.2
25), and CA at 3 mg/d (CA3, n = 26). Treatment   Mycotoxin binder3 0.5
CH1 represents what is typically used as vitamin D   Mineral and vitamin premix4 4.2
Nutrient content (DM basis; mean ± SD)  
feeding to dairy cows in commercial farms (Nelson et  NEL,5 Mcal/kg 1.48
al., 2016), and it is twice the minimum amount cur-   OM, % 91.0 ± 0.5
rently recommended by NRC (2001) for prepartum   CP, % 13.9 ± 0.1
  NDF, % 40.9 ± 1.9
cows. The different numbers of experimental units per  NFC,6 % 36.4 ± 2.7
treatment was caused by some cows calving before the   Ether extract, % 3.2 ± 0.4
expected date, and, therefore, a prepartum sample was   Ca, % 0.53 ± 0.05
 P, % 0.29 ± 0.01
not obtained, resulting in incomplete blocks. Treat-   Mg, % 0.44 ± 0.01
ments were initiated at 250 d of gestation, and vitamin   K, % 1.27 ± 0.05
D supplements were diluted into 100 g of corn meal   S, % 0.41 ± 0.03
  Na, % 0.18 ± 0.01
to result in the desired doses of CH (1 or 3 mg) and   Cl, % 0.80 ± 0.04
CA (1 or 3 mg). Cows were fed the same prepartum  DCAD,7 mEq/kg −77 ± 34
diet offered as TMR once daily, at 0700 h, which was 1
Prepartum diet was fed from 250 d of gestation to calving.
formulated to meet the nutrient needs of late-gestation 2
Bio-Chlor (Arm and Hammer Animal Nutrition): fermentation prod-
Holstein cows and formulated to have a negative value uct containing dried condensed extracted glutamic acid fermentation
product, dried condensed corn fermentation solubles, processed grain
for DCAD (Table 1). The vitamin D treatments were by-products, and magnesium chloride.
administered as top-dress dispensed onto the TMR 3
Novasil Plus (BASF Corp.).
once daily until calving. Postpartum, cows were fed the 4
Contained (DM basis) 52.2% wheat middlings, 18.7% Reashure
same diet containing 1 mg of CH for every 25 kg of DM. (Balchem), 16.0% magnesium sulfate heptahydrate, 4.8% magnesium
oxide, 4.5% sodium chloride, 0.685% of a mixture containing vitamins
A, D, and E and iodine, 0.45% Sel-Plex 2000 (Alltech Biotechnology),
Plasma Concentrations of Vitamin D 0.40% Rumensin 90 (Elanco Animal Health), 0.27% IntelliBond Vital4
(Micronutrients), and 2.0% ClariFly Larvicide (Central Life Sciences).
Each kilogram contained 8.8% CP, 0.20% Ca, 0.59% P, 4.4% Mg, 0.5%
Blood was sampled by puncture of the coccygeal ves- K, 1.8% Na, 2.7% Cl, 2.2% S, 740 mg of Zn, 168 mg of Cu, 562 mg
sels into 10-mL evacuated tubes containing K2 EDTA of Mn, 9 mg of Se, 16.2 mg of Co, 12 mg of I, 103,000 IU of vitamin
(Vacutainer, Becton Dickinson). A sample was collected A, 18,160 IU of vitamin D, 1,475 IU of vitamin E, and 725 mg of
monensin.
the day before cows initiated the dietary treatments 5
Calculated using NRC (2001) according to the chemical composition
and again on d 260, 267, 273, and 275 of gestation, of the dietary ingredients and adjusted for 11 and 20 kg of DMI for the
which corresponded to the mean days relative to calving pre- and postpartum periods, respectively.
(range) −19 (−23 to −14), −10 (−13 to −4), −3 (−5 6
Calculated as follows: NFC = DM − (ash + CP + ether extract +
to −2) and −1 (−1). Postpartum, blood was sampled NDF − NDF-insoluble CP).
7
Calculated as follows: DCAD = [(mEq of K) + (mEq Na)] − [(mEq
exactly on d 0, 4, and 8. Concentrations of vitamin D3, of Cl) + (mEq of S)].
25-hydroxyvitamin D3, and 24,25-dihydroxyvitamin D3
in plasma were analyzed using ultra high-performance
liquid chromatography (1290 Infinity II LC System, ng/mL for vitamin D3 and 24,25-hydroxyvitamin D3,
Agilent) coupled with tandem MS detection (API and 1.0 ng/mL for 25-hydroxyvitamin D3. The intra-
4000 LC-MS/MS System, AB Sciex LLC) by the DSM assay coefficients of variation (CV) were 4.6, 3.6, and
Nutritional Products Research and Development Solu- 6.2%, respectively, for vitamin D3, 25-hydroxyvitamin
tion Center (Kaiseraugst, Switzerland). In all assays, D3, and 24,25-dihydroxyvitamin D3.
dedicated standard and quality-control samples were
analyzed concurrent with unknown samples to ensure Blood Minerals and Metabolites
the accuracy and precision of the method. Data acqui-
sition, integration, and quantification were performed Blood was sampled by puncture of the coccygeal
using Analyst 1.7 software (AB Sciex LLC). Personnel vessels into 10-mL serum separator evacuated tubes
running the assays were blind to treatments, and the (Vacutainer, Becton Dickinson) on gestation d 250,
lower limits of quantification for the assays were 0.5 before treatments initiated, and again on d 260, 263,
Journal of Dairy Science Vol. 104 No. 10, 2021
 Vieira-Neto et al.: SOURCE AND AMOUNT OF VITAMIN D AND IMMUNE CELLS
266, and 269 of gestation, and then daily until calving.
Postpartum blood was sampled on the day of calving
and again on d 1 to 5 postpartum. Samples were col-
lected before the morning feeding at 0600 h. Tubes were
placed on ice, transported to the laboratory within 1
h, and centrifuged for 20 min at 2,500 × g for serum
separation. Serum was frozen and stored at −20°C for
further analyses. Prepartum serum samples collected
from d −9 to −1 relative to calving and daily from d
0 to 5 postpartum were assayed for concentrations of
total Ca, Mg, and P. Serum was analyzed for concen-
trations of Ca (Arsenazo III method, cat. no. CA2390),
Mg (xylidyl-blue method, cat. no. MG531), and P
(phosphomolybdate method, cat. no. PH8328) using a
biochemical analyzer (RX Daytona, Randox Laborato-
ries Ltd.) according to the manufacturer’s instructions.
Intra- and interassay CV were, respectively, 0.5% and
8.1% for Ca, 4.1% and 10.2% for Mg, and 1.1% and
9.8% for P.
Peripheral Blood Leukocyte Population Markers
Leukocytes were isolated from coccygeal blood sam-
pled into 10-mL lithium-heparinized tubes (Vacutainer,
Becton Dickinson) prepartum at 270 d of gestation, and
on d 3 and 6 postpartum. Erythrocytes were lysed from
an aliquot containing 100 μL of whole blood by adding
2 mL of a lysing solution (10.6 mM Na2HPO4, 2.7 mM
NaH2PO4, pH = 7.2) and keeping tubes on ice for 2
min. Then 1 mL of a resuspension solution (10.6 mM
Na2HPO4, 2.7 mM NaH2PO4, 462 mM NaCl, pH = 7.2)
was added, and samples were homogenized and cen-
trifuged at 500 × g for 5 min at 4°C. The supernatant
was removed, and a small pellet of cells remained in the
bottom of the tube. Cells were incubated for 20 min on
ice with allophycocyanin (APC, Bio-Rad Laboratories)-
conjugated anti-CD11b (CC126 clone, Bio-Rad Labo-
ratories), fluorescein isothiocyanate (FITC)-conjugated
anti-CD21 (CC21 clone, Bio-Rad Laboratories), phyco-
erythrin (PE, Bio-Rad Laboratories)-conjugated anti-
CD62L (BAQ92A clone, Kingfisher Biotech Inc.), and
phycoerythrin and cyanine dye (PE-Cy5.5) anti-CD14
(Tük4 clone, Life Technologies). An antibody cocktail
solution was prepared containing 25 μL of each anti-
body for a total of 100 μL (1 μg/μL), which was suf-
ficient for 100 samples, and stored at 4°C in the dark.
On the day of the assay, 1 μL of antibody cocktail was
diluted into 25 μL of flow staining buffer (PBS with 5%
heat-inactivated fetal bovine serum and 0.1% NaN3) in
a microtube kept in the dark and added to each pellet
of cells isolated from a cow and incubated on ice for
15 min. Cells were washed with 2 mL of flow staining
buffer to remove excess of antibodies not bound and
then centrifuged at 500 × g for 5 min at 4°C. Cells were
Journal of Dairy Science Vol. 104 No. 10, 2021
resuspended in 500 μL of cold PBS at 4°C and kept on
ice in the dark until analysis.
Cell populations were then analyzed by flow cytom-
etry (Accuri C6 digital analyzer flow cytometer, BD
Biosciences) according to forward and side scatters to
segregate cells based on size and granularity, and then
identify those expressing CD14, CD21, CD11b, and
CD62L. Data acquisitions from 10 μL of resuspended
cells per sample (16,924 ± 13,881 cells) were analyzed
using FlowJo software (version 10.6.2, FlowJo LLC).
The parameters analyzed included the percentage of
granulocytes and mononuclear cells from the total
leukocyte population, and percentages of monocytes
(CD14+), B lymphocytes (CD14− CD21+), and CD14−
CD21− cells from the population of mononuclear cells
(Supplemental Figure S1A; https:​/​/​figshare​.com/​s/​
f19a05c4324597477f6e). We were limited to 4 param-
eters in the flow cytometry panel, and these markers
were selected to most effectively capture the profile
and status of circulating leukocytes. After identifica-
tion of the distinct cell populations, the mean fluores-
cence intensity (MFI) for specific cell surface markers
was quantified. In granulocytes, MFI was quantified
for L-selectin (CD62L) and a β-integrin (CD11b); in
monocytes, MFI was quantified for CD14, CD62L, and
CD11b; in B lymphocytes, the MFI was quantified for
CD62L and CD11b; and in CD14− CD21− cells, the
MFI was quantified for CD62L and CD11b.
Granulocytes Function
Blood was sampled by puncture of the coccygeal ves-
sels into 10-mL lithium-heparinized tubes (Vacutainer,
Becton Dickinson) at 270 d of gestation and on d 3 and
6 postpartum. Tubes were kept at room temperature,
wrapped in foil to protect from light, and transported
to the laboratory within 3 h of collection. Granulocyte
function was assayed in vitro by dual-color flow cy-
tometry according to Vieira-Neto et al. (2017) with
a few modifications. Quantification of granulocyte
phagocytosis and intracellular killing was assessed us-
ing an Escherichia coli 08:H19 strain KCJ852 previ-
ously isolated from a cow with metritis, labeled with
propidium iodide. Briefly, 100 μL of blood was pipetted
in triplicate aliquots, and 10 μL of 5 μM dihydrorho-
damine 123 was added to each aliquot. One aliquot
was used as a negative control, containing only blood
and 5 μM dihydrorhodamine 123. The other 2 aliquots
each received 40 μL of a solution containing 5.0 × 105
propidium iodide-labeled E. coli per μL. Samples were
analyzed by flow cytometry (BD Biosciences). Data ac-
quisitions from at least 10,000 and up to 100,000 cells
per sample were analyzed using FlowJo software. Cells
were then analyzed according to forward and side scat-
 Vieira-Neto et al.: SOURCE AND AMOUNT OF VITAMIN D AND IMMUNE CELLS
ter gating, and responses analyzed included percentage
of granulocytes that phagocytized bacteria, percentage
of granulocytes with phagocytosis-induced oxidative
burst, MFI for phagocytosis (an indication of the num-
ber of bacteria phagocytized per granulocyte), and MFI
of oxidized dihydrorhodamine to estimate intensity of
oxidative burst as an indication of reactive oxygen spe-
cies produced per cell.
Leukocyte RNA Extraction and Gene Expression
Blood leukocytes were isolated from samples col-
lected at 270 d of gestation and on d 3 postpartum.
Blood was sampled by puncture of the coccygeal vessels
into 10-mL lithium-heparinized tubes (Vacutainer, Bec-
ton Dickinson). Samples were centrifuged for 20 min
at 2,500 × g for plasma separation, and the buffy coat
fraction was collected by pipetting and transferring to
a 12-mL conical tube. Red blood cell lysis buffer (Cell
Lysis Solution, Promega Corp.) was added for a total
volume of 12 mL. Tubes were homogenized and incu-
bated at room temperature for 5 min. Samples were
then centrifuged for 5 min at 500 × g and supernatant
discarded. This step was repeated multiple times to
remove all red blood cells. The leukocyte pellets were
resuspended with 0.8 mL of Trizol (TRIzol LS Re-
agent, Invitrogen), transferred to 1.5-mL microtubes,
and stored at −80°C until further analysis. For RNA
extraction, chloroform was added to the microtube to
bring up to a 20% chloroform solution. Samples were
then centrifuged at 12,000 × g for 15 min at 4°C, and
the supernatant containing the RNA was transferred to
a new microtube. Purification of RNA was performed
using the PureLink RNA Mini Kit (Invitrogen) ac-
cording to the manufacturer’s instruction. Purity and
concentration were evaluated using a NanoDrop 2000
spectrophotometer (Thermo Scientific). Samples had
a mean (±SD) 260:280 nm ratio of 2.00 ± 0.06, and
260:230 nm ratio of 1.75 ± 0.39.
The mRNA for a selected set of genes was quantified
by the Fluidigm quantitative PCR microfluidic device
Biomark HD system (Fluidigm Co.). The PCR primers
were designed by Fluidigm Delta Gene assays and syn-
thesized by Fluidigm (Fluidigm Co.). Details of genes
and primers are in Supplemental Table S1 (https:​/​/​
figshare​.com/​s/​f19a05c4324597477f6e). A pool sample
containing mRNA from bovine leukocytes from 10 dif-
ferent samples was used for primer validation. Primers
were validated using Fluidigm primer quality-control
criteria (R2 ≥ 0.97; efficiency of 80 to 130%; slope =
−3.92 to −2.76) were applied to cDNA serially diluted
by 12× and evaluated in 8 replicates. Primers targeting
8 genes (CASR, CYP24A1, GC, IL12A, MMP2, NOS2,
SELE, and TRPV6) failed to pass the quality control in
Journal of Dairy Science Vol. 104 No. 10, 2021
the qualification run and were excluded from analyses.
Primer efficiency in the genes passing quality control
and used in this experiment ranged between 101.3 and
120% (Supplemental Table S1; https:​/​/​figshare​.com/​
s/​f19a05c4324597477f6e). Genes investigated included
those involved in cell adhesion and migration, patho-
gen recognition receptor, cell signaling, synthesis of
cytokines, antimicrobial mechanisms, oxidative burst,
Ca metabolism, and vitamin D metabolism. Statistical
analyses were performed on dCt values as described
by Steibel et al. (2009). Fold changes relative to a
reference treatment were calculated using the method
described by Yuan et al. (2006), whereby fold changes
were calculated from least squares means (LSM) dif-
ference according to the formula 2−ddCt, where dCt =
CtTarget gene − geometric mean of CtReference genes, and ddCt
= dCttreatment A − dCttreatment B. Reference genes used
were ACTB, GAPDH, RPL19, RPS9, and YWHAZ.
Heatmaps were generated using Heatmapper online
tool (Babicki et al., 2016).
Statistical Analyses
Distribution of residuals and homogeneity of variance
were evaluated for all continuous dependent variables
after fitting the statistical models. Responses that vio-
lated the assumptions of normality were subjected to
power transformation according to the Box-Cox pro-
cedure (Box and Cox, 1964) using a macro for mixed
models in SAS (SAS STAT version 9.4, SAS Institute
Inc.) according to Piepho (2009). Concentrations in
plasma of vitamin D3, 25-hydroxyvitamin D3, and
24,25-dihydroxyvitamin D3 had to be log-transformed
before analysis either because of heteroscedasticity or
because residuals were not normally distributed. The
LSM and standard error of the mean (SEM) were
back-transformed for data presentation according to
Jørgensen and Pedersen (1998).
Data were analyzed by linear mixed-effects models
using the MIXED procedure of SAS, and analyses were
performed for the pre- and postpartum periods sepa-
rately. The statistical models included the fixed effects
of source of vitamin D (CH vs. CA), amount of vita-
min D [1 mg/d (1mg) vs. 3 mg/d (3mg)], interaction
between source and amount (CH1 + CA3 vs. CH3 +
CA1), the linear covariate of the exact day relative to
calving when the measurement was taken, and the ran-
dom effect of block. For analysis of mRNA expression,
the fixed effect of assay plate (1 or 2) was also included
in the models. Data with repeated measures within
cow were analyzed with the same mixed-effects model
described previously but also included the fixed effects
of day and the interactions between source of vitamin
D and day, amount of vitamin D and day, and source
 Vieira-Neto et al.: SOURCE AND AMOUNT OF VITAMIN D AND IMMUNE CELLS
and amount and day, as well as the random effect of
cow nested within source and amount of vitamin D.
For concentrations of vitamin D metabolites in plasma,
the concentration obtained in the sample analyzed
before initiation of treatments was used as covariate
in the statistical models. The Repeated statement was
included in all mixed models with repeated measure-
ments, with day specified as the repeated effect. The
covariance structure was selected based on spacing be-
tween measurements and model fit that resulted in the
smallest corrected Akaike’s information criterion. The
Kenward-Roger method was used to approximate de-
nominator degrees of freedom to compute the F tests.
When an interaction between amount and source of
supplemental vitamin D was significant, means were
portioned using the SLICE command in SAS, and
pairwise comparisons were conducted by the Fisher’s
least significant difference. Evidence of statistical sig-
nificance against the null hypothesis was considered at
P ≤ 0.05, and tendency was considered at 0.05 < P ≤
0.10. For gene expression, only comparisons with P <
0.05 were considered.
RESULTS
Prepartum Vitamin Intake and Blood Concentrations
of Vitamins and Minerals
The DMI prepartum did not differ with source or
amount of vitamin D and averaged 11.3 kg/d. The diet
offered contained some CH in the mineral-vitamin pre-
mix, which resulted in a mean prepartum intake of CH
from the diet of 0.22 mg/d in all treatments. Therefore,
cows consumed daily 1.22 mg of CH in CH1, 3.22 mg
of CH in CH3, 0.22 mg of CH and 1 mg of CA in
CA1, and 0.22 mg of CH and 3 mg of CA in CA3. The
average duration of treatment prepartum did not dif-
fer among treatments, and means were 24.2 (9 to 32),
25.7 (13 to 33), 25.1 (14 to 32), and 25.5 (18 to 30) d,
respectively, for CH1, CH3, CA1, and CA3. Six cows
developed milk fever, 2 out of 21 in CH1 (9.5%), 1 out
of 27 in CH3 (3.7%), 1 out 25 in CA1 (4.0%), and 2 out
of 26 in CA3 (7.7%).
Of the 99 cows enrolled, 25 cows with 66 samples
had concentration of vitamin D3 in plasma below the
detection limit of the assay (<0.50 ng/mL). Of those
25 cows, 3 cows were fed CH, 2 in CH1 and in 1 CH3,
and 22 cows were fed CA, 12 in CA1 and 10 in CA3.
Details of the number of samples, day relative to calv-
ing, and number of cows with concentrations below the
detection limit for vitamin D3 in plasma are presented
in Supplemental Table S2 (https:​/​/​figshare​.com/​s/​
f19a05c4324597477f6e). All other measurements of
vitamin D3, 25-hydroxyvitamin D3, and 24,25-dihy-
Journal of Dairy Science Vol. 104 No. 10, 2021
droxyvitamin D3 resulted in concentrations above the
detection limits of the assays.
All means and measures of variance reported in the
text are LSM and respective SEM, unless otherwise
stated. Concentrations of vitamin D3 in plasma prepar-
tum decreased (P < 0.001) as parturition approached,
in particular in cows fed CH3 (Figure 1A). Concentra-
tions decreased (P < 0.001) over time postpartum, and
the decline was observed only in cows fed CH based
on the interaction (P < 0.001) between source and
day relative to calving. However, feeding CA increased
(P < 0.001) concentrations of 25-hydroxyvitamin D3
in plasma, and the increment over time was greater
in cows fed CA3 than CA1 (Figure 1B). Concentra-
tions of 25-hydroxyvitamin D3 in plasma declined (P <
0.001) in the first 8 d postpartum, and an interaction
(P = 0.04) among source and amount and day was
observed because the decrease in concentrations was
greater in cows fed CA3 than in all other treatments.
An interaction (P < 0.001) among source and amount
and day was observed for prepartum concentrations
of 24,25-hydroxyvitamin D3 in plasma because of the
greater increase in concentrations in CA3 compared
with all other treatments (Figure 1C). Concentrations
of 24,25-hydroxyvitamin D3 in plasma remained el-
evated postpartum in cows fed CA3, followed by CA1,
and then those fed CH.
Cows fed CA had greater (P = 0.03) concentrations
of P in serum prepartum than those fed CH (CH =
1.84 vs. CA = 1.93 ± 0.03 mM; Table 2). Nevertheless,
treatment or the interaction between treatment and
day did not affect the concentrations of Ca or Mg in
serum prepartum or any of the minerals postpartum.
Incidence of Diseases
Uterine diseases, defined as the combination of re-
tained placenta and metritis, affected 22.2% (22/99)
of the cows in this experiment; however, no difference
between treatments was detectable (CH1 = 28.4; CH3
= 18.3; CA1 = 20.5; CA3 = 22.8 ± 8.6%). Mastitis
affected 7.1% (7/99) of the cows and, similar to uterine
diseases, was not affected by source or amount of vi-
tamin D (CH1 = 3.8; CH3 = 13.3; CA1 = 3.5; CA3 =
3.3 ± 4.5%). During the first 60 d postpartum, 36.4%
(36/99) had at least 1 disease event; however, no differ-
ence was detectable between treatments (CH1 = 47.5;
CH3 = 36.9; CA1 = 29.1; CA3 = 34.5 ± 9.8%).
Profile of Immune Cells in Blood
Feeding the 3mg treatments, irrespective of source
of vitamin D, increased (P = 0.008) the proportion of
granulocytes (1mg = 24.5 vs. 3mg = 37.9 ± 3.5%) but
 Vieira-Neto et al.: SOURCE AND AMOUNT OF VITAMIN D AND IMMUNE CELLS

Figure 1. Concentrations of vitamin D3 (A), 25-hydroxyviatmin D3 (B), and 24,25 dihydroxyvitamin D3 (C) in plasma of dairy cows supple-
mented with 1 or 3 mg of cholecalciferol (CH1 or CH3) or calcidiol (CA1 or CA3) from 250 d of gestation to calving. Cov = covariate value
based on sample collected before starting treatments. Error bars depict SEM. Panel A, prepartum: effect of source (P < 0.001), amount (P <
0.001), and interaction between source and amount (P < 0.001); postpartum: effect of source (P < 0.001), amount (P < 0.001), and interaction
between source and amount (P < 0.001). Panel B, prepartum: effect of source (P < 0.001), amount (P < 0.001), and interaction between source
and amount (P < 0.01); postpartum: effect of source (P < 0.001), amount (P < 0.001), and interaction between source and amount (P < 0.001).
Panel C, prepartum: effect of source (P < 0.001), amount (P < 0.001), and interaction between source and amount (P < 0.001); postpartum:
effect of source (P < 0.001), amount (P < 0.001), and interaction between source and amount (P < 0.001).

consequently decreased that of mononuclear cells (1mg
= 75.5 vs. 3mg = 62.1 ± 3.5%) in blood prepartum
(Figures 2A and 2B). Source of vitamin D did not af-
fect the proportions of different leukocytes in blood.
Similar responses were observed during the early post-
partum period. Feeding the 3mg treatments increased
(P = 0.02) the proportion of granulocytes (1mg = 22.0
vs. 3mg = 31.0 ± 3.0%) and decreased that of mono-
nuclear cells (1mg = 78.0 vs. 3mg = 69.0 ± 3.0%) in
blood compared with feeding 1mg treatments (Figures
2A and 2B). Cows fed CA tended (P = 0.08) to have
a smaller proportion of granulocytes (CH = 29.8 vs.
CA = 23.3 ± 3.0%) but a greater proportion of mono-
nuclear cells (CH = 70.2 vs. CA = 76.7 ± 3.0%) in
blood than cows supplemented with CH. No interaction
was observed between source and amount of vitamin
D pre- or postpartum for the proportion of different
leukocytes in blood.
Regarding mononuclear cells, feeding CH3 increased
(P < 0.05) the proportion of monocytes pre- and post-
partum (Figure 2C). Consequently, cows fed CH3 had
the smallest proportions of B lymphocytes and CD14−

Table 2. Serum concentrations of minerals pre- and postpartum (LSM ± SEM)1

CH CA P-value2

Item 1 mg 3 mg   1 mg 3 mg SEM Source Amount Source × amount

3
Prepartum                  
 Ca 2.30 2.26   2.28 2.28 0.02 0.98 0.36 0.35
 Mg 0.83 0.79   0.80 0.79 0.02 0.36 0.14 0.43
 P 1.85 1.84   1.91 1.96 0.04 0.03 0.55 0.42
Postpartum4                  
 Ca 2.05 2.04   2.07 2.03 0.03 0.96 0.40 0.69
 Mg 0.89 0.88   0.89 0.85 0.02 0.47 0.22 0.43
 P 1.75 1.77   1.79 1.73 0.05 0.94 0.72 0.45
1
Prepartum cows at 250 d of gestation were supplemented with 1 or 3 mg of either cholecalciferol (CH) or calcidiol (CA).
2
Source = effect of source of vitamin D (CH vs. CA); amount = effect of amount of vitamin D (1 vs. 3 mg); source × amount = interaction
between source and amount (CH1 + CA3 vs. CH3 + CA1).
3
Concentrations (mM) of Ca, Mg, and P were determined in serum samples collected daily from d −9 to −1 prepartum.
4
Concentrations (mM) of Ca, Mg, and P were determined in serum samples collected daily from d 0 to 5 postpartum.

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 Vieira-Neto et al.: SOURCE AND AMOUNT OF VITAMIN D AND IMMUNE CELLS

CD21− cells (Figure 2D). Cows fed 3 mg of either
source of vitamin D had the smallest proportions of
CD14− CD21− cells pre- and postpartum (Figure 2E).
Feeding CA decreased (P = 0.05) the percentage of
granulocytes expressing CD62L compared with CH
prepartum (Table 3). In addition, MFI for CD62L was
also less (P = 0.03) for granulocytes from cows fed CA
compared with CH. Treatment did not affect the MFI
for CD11b on granulocytes. On monocytes, MFI for
CD14 was less (P = 0.02) in cows fed CA compared
with CH, and those fed 3mg had greater (P = 0.02)
MFI for CD14 compared with 1mg. Treatment did not
affect the percentage of monocytes expressing CD62L
and MFI for CD62L on those cells. Cows fed CA tended
(P = 0.06) to have greater MFI for CD11b on mono-
cytes than cows fed CH.
A tendency for interaction (P = 0.09) between source
and amount of vitamin D was observed for the percent-
age of B lymphocytes expressing CD62L prepartum,
because feeding CA1 tended to have greater percent-
age of cells expressing CD62L compared with those fed
CA3. Also, MFI for CD62L in B lymphocytes increased
(P < 0.05) with feeding CA1 compared with CA3.
Treatment did not affect MFI for CD11b in B lym-
phocytes. For CD14− CD21− cells prepartum, feeding
the 3mg treatments tended (P = 0.07) to increase the
percentage of cells expressing CD62L compared with
the 1mg treatments (1mg = 12.0 vs. 3mg = 18.4 ±
2.5%). Similarly, cows fed 3mg treatments tended (P
= 0.09) to have increased MFI for CD62L in CD14−
CD21− cells prepartum compared with those fed 1mg
treatments (1mg = 2.35 vs. 3mg = 2.44 ± 0.04).
Treatment did not affect percentage of granulocytes
expressing CD62L, or MFI for CD62L and CD11b in
granulocytes postpartum (Table 4). In cows fed CH3,
MFI for CD14 was increased (P < 0.05) compared with
the other treatments. Percentage of monocytes express-
ing CD62L increased (P = 0.04) in cows fed CH com-

Figure 2. Percentage of granulocytes (A) and mononuclear cells (B) from total leukocytes, and percentage of monocytes (C), B lymphocytes
(D), and CD14− CD21− cells (E) from the mononuclear cells. Prepartum cows were supplemented with 1 or 3 mg of cholecalciferol (CH1 or CH3)
or calcidiol (CA1 or CA3) from 250 d of gestation to calving. Error bars depict SEM. Panel A, prepartum: effect of source (P = 0.30), amount (P
= 0.008), and interaction between source and amount (P = 0.44); postpartum: effect of source (P = 0.08), amount (P = 0.02), and interaction
between source and amount (P = 0.18). Panel B, prepartum: effect of source (P = 0.30), amount (P = 0.008), and interaction between source and
amount (P = 0.44); postpartum: effect of source (P = 0.08), amount (P = 0.02), and interaction between source and amount (P = 0.18). Panel C,
prepartum: effect of source (P = 0.37), amount (P = 0.02), and interaction between source and amount (P = 0.04); postpartum: effect of source
(P = 0.15), amount (P = 0.05), and interaction between source and amount (P = 0.18). Panel D, prepartum: effect of source (P = 0.78), amount
(P = 0.65), and interaction between source and amount (P = 0.07); postpartum: effect of source (P = 0.55), amount (P = 0.84), and interaction
between source and amount (P = 0.49). Panel E, prepartum: effect of source (P = 0.43), amount (P = 0.02), and interaction between source
and amount (P = 0.78); postpartum: effect of source (P = 0.33), amount (P = 0.07), and interaction between source and amount (P = 0.45).

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 Vieira-Neto et al.: SOURCE AND AMOUNT OF VITAMIN D AND IMMUNE CELLS

pared with CA. Similarly, the MFI for CD62L increased
(P = 0.02) in cows fed CH compared with CA. Within
cows fed CA, 3mg reduced (P < 0.05) the MFI for
CD11b in monocytes compared with 1mg, whereas no
difference occurred among cows fed CH. In B lympho-
cytes, the percentage of cells expressing CD62L did not
differ among treatments, whereas cows fed CH3 had
greater (P < 0.05) MFI for CD62L compared with all
other treatments. No difference was detectable between
treatments for the MFI for CD11b in B lymphocytes.
In CD14− CD21− cells, feeding 3mg increased (P <
0.05) the percentage of cells expressing CD62L (1mg =
11.0 vs. 3mg = 16.4 ± 2.2%) and MFI of CD62L (1mg
= 2.35 vs. 3mg = 2.45 ± 0.04) but decreased (P < 0.05)
the MFI of CD11b (1mg = 3.32 vs. 3mg = 3.19 ± 0.06)
compared with those fed 1mg.
Granulocyte Function
The proportion of granulocytes with phagocytic ac-
tivity prepartum tended (P = 0.06) to be less in cows fed
3mg compared with 1mg (1mg = 69.0 vs. 3mg = 62.9
± 3.0%), whereas treatment did not affect granulocyte
phagocytosis postpartum (Figure 3A). Treatment did
not affect the proportion of granulocytes with oxida-
tive burst pre- or postpartum (Figure 3B). Intensity
of phagocytosis prepartum tended to be smaller (P =
0.06) for cows fed 3mg compared with 1mg (1mg = 7.46
vs. 3mg = 7.28 ± 0.09).
No effect of treatment was observed for MFI of
phagocytosis postpartum (Figure 3C). An interaction
(P = 0.05) between source and amount of vitamin D
was observed for MFI of oxidative burst prepartum,
in part because of a tendency (P = 0.07) for CA1 to
be greater than CH1 (Figure 2D). Treatment did not
affect MFI for oxidative burst postpartum.
mRNA Expression in Blood Leukocytes Prepartum
A heatmap with the genes that were differentially
expressed during the prepartum period was created
using the fold-change relative to CH1 to illustrate
the patterns according to treatment (Figure 4A). In
addition, Supplemental Figure S2A depicts the pat-
tern of mRNA expression according to the dCt for
individual cows including only genes that were influ-
enced (P < 0.05) by treatment (https:​/​/​figshare​.com/​
s/​f19a05c4324597477f6e). In both Figure 4A and
Supplemental Figure S2A (https:​/​/​figshare​.com/​s/​
f19a05c4324597477f6e), red represents reduced mRNA
expression, whereas green represents increased mRNA
expression. The genes differentially expressed (P <

Table 3. Effect of source and amount of vitamin D on blood cell markers prepartum (LSM ± SEM)1

CH CA P-value2

Item 1 mg 3 mg   1 mg 3 mg SEM Source Amount Source × amount

3
Granulocytes                  
 CD62L+, % 77.7 74.5   69.5 61.9 5.6 0.05 0.29 0.66
  CD62L, MFI4 log10 3.59 3.54   3.48 3.43 0.06 0.03 0.35 0.96
  CD11b, MFI log10 3.46 3.55   3.44 3.46 0.06 0.17 0.16 0.33
Monocytes5                  
  CD14, MFI log10 4.38 4.59   4.26 4.38 0.07 0.02 0.02 0.49
 CD62L+, % 53.2 52.6   48.4 44.7 4.9 0.13 0.59 0.70
  CD62L, MFI log10 2.46 2.27   2.33 2.07 0.35 0.61 0.49 0.90
  CD11b, MFI log10 3.53 3.65   3.52 3.54 0.05 0.13 0.06 0.16
B lymphocytes5                  
 CD62L+, % 9.1 11.8   13.3A 7.9B 2.5 0.95 0.58 0.09
  CD62L, MFI log10 2.35A 2.41   2.45a,B 2.33b 0.05 0.78 0.45 0.03
  CD11b, MFI log10 3.30 3.31   3.22 3.36 0.05 0.74 0.12 0.18
CD14− CD21− cells                  
 CD62L+, % 11.9 21.3   12.0 15.5 3.5 0.42 0.07 0.39
  CD62L, MFI log10 2.34 2.48   2.36 2.41 0.06 0.65 0.09 0.43
  CD11b, MFI log10 3.28 3.13   3.28 3.25 0.07 0.38 0.19 0.34
a,b
Values within a row with different lowercase superscripts differ (P < 0.05).
A,B
Values within a row with different uppercase superscripts differ (0.05 < P < 0.10).
1
Prepartum cows at 250 d of gestation were supplemented with 1 or 3 mg of cholecalciferol (CH) or calcidiol (CA). Blood was sampled and
analyzed on d 270 of gestation.
2
Source = effect of source of vitamin D (CH vs. CA); amount = effect of amount of vitamin D (1 vs. 3 mg); source × amount = interaction
between source and amount (CH1 + CA3 vs. CH3 + CA1).
3
Identified by flow cytometry according to size and granularity.
4
MFI = mean fluorescence intensity.
5
Identified by flow cytometry based on expression of cell surface markers.

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 Vieira-Neto et al.: SOURCE AND AMOUNT OF VITAMIN D AND IMMUNE CELLS

0.05) by treatment during the prepartum period are
reported in Table 5. Those included genes related to
cell adhesion and migration, CD44, ICAM1, ITGAL,
ITGB1, LGALS8, and SELL, which were upregulated
(P < 0.05) by CA compared with CH. Expression of
CXCR2, ITGAM, ITGB2, and TLN1 did not differ
among treatments. Expression of genes related to
receptors for pathogen recognition, including NOD2,
TLR2, and TLR6, were upregulated (P < 0.05) in cows
fed CA compared with CH. By contrast, expression of
NOD1 was downregulated (P = 0.01) in cows fed CA
compared with CH. Expression of TLR1, TLR4, and
TLR9 did not differ among treatments. Expression of
genes related to cell signaling cascade, including FOS,
JUN, and NFKB2, were upregulated (P < 0.05) by
CA compared with CH, whereas expression of AKT1,
AKT2, IRAK4, MAPK1, MAPK3, MYD88, NFATC1,
and NFKB1 did not differ among treatments. Calcidiol
upregulated (P < 0.05) genes involved in cytokine sig-
naling, including IL1B, IL1R1, and IL1RN, whereas
expression of CCL2, CCL5, CXCL8, IFNG, IL1R2,
IL6, IL10, IL23A, and TNF did not differ among
treatments. Calcidiol upregulated (P < 0.05) genes in-
volved in antimicrobial activity, including CTSB and
LYZ, whereas expression of BPI, CATHL5, CATHL6,
DEFB1, DEFB10, DEFB3, DEFB4A, DEFB5,
DEFB6, DEFB7, ELANE, LAP, LTF, PRKCB, and
TAP did not differ among treatments. In addition,
treatment did not affect genes related to oxidative
burst, including CYBA, CYBB, GPX1, GSR, MPO,
NCF1, NCF2, NCF4, RAC2, and SOD1. Calcidiol
upregulated (P < 0.05) genes involved in Ca binding
and transport, including ATP2B1 and STIM1. Cows
fed 3mg had less (P = 0.01) expression of TRPV5
than those fed 1mg. Expression of CALM1, CALM2,
CALM3, ITPR1, ORAI1, PPP3CA, PPP3CB, and
SLC8A1 did not differ among treatments. In addition,
expression of genes related to vitamin D metabolism,
including CYP27B1, RXRA, and VDR, did not differ
among treatments.
mRNA Expression in Blood Leukocytes Postpartum
The pattern of expression of genes in leukocytes
postpartum affected (P < 0.05) by treatment are
depicted in the heatmap in Figure 4B. The same
heatmap is depicted for individual cows postpartum
in Supplemental Figure S2B (https:​/​/​figshare​.com/​
s/​f19a05c4324597477f6e). Calcidiol upregulated (P <
0.05) genes involved in cell adhesion and migration,
including CXCR2, SELL, and TLN1; cell signaling cas-
cade, AKT2; synthesis of cytokines, including CCL2,
IL1R1, and IL1RN; and antimicrobial mechanisms,
DEFB3 and RAC2; but it downregulated (P < 0.05) a

Table 4. Effect of source and amount of vitamin D on blood cell markers postpartum (LSM ± SEM)1

CH CA P-value2

Item 1 mg 3 mg   1 mg 3 mg SEM Source Amount Source × amount

3
Granulocytes                  
 CD62L+, %4 78.5 73.8   72.1 69.2 4.6 0.18 0.36 0.83
  CD62L, MFI5 log10 3.64 3.44   3.46 3.46 0.09 0.37 0.25 0.28
  CD11b, MFI log10 3.56 3.51   3.54 3.48 0.07 0.60 0.33 0.91
Monocytes6                  
  CD14, MFI log10 4.39a 4.61b   4.40a 4.40a 0.07 0.07 0.04 0.06
 CD62L+, % 40.4 40.7   34.7 36.0 3.4 0.04 0.75 0.84
  CD62L, MFI log10 2.01 1.77   1.44 1.45 0.24 0.02 0.55 0.53
  CD11b, MFI log10 3.57ac 3.66bc   3.63c 3.56a 0.06 0.58 0.63 0.004
B lymphocytes6                  
 CD62L+, % 7.8 11.1   9.2 7.4 2.4 0.58 0.71 0.22
  CD62L, MFI log10 2.32 2.47   2.33 2.30 0.07 0.13 0.20 0.07
  CD11b, MFI log10 3.30 3.24   3.29 3.27 0.06 0.82 0.43 0.68
CD14− CD21− cells                  
 CD62L+, % 11.8 18.8   10.1 14.0 3.0 0.24 0.05 0.57
  CD62L, MFI log10 2.36 2.48   2.34 2.42 0.05 0.37 0.03 0.68
  CD11b, MFI log10 3.26 3.15   3.38 3.22 0.07 0.13 0.03 0.64
a–c
Values within a row with different superscripts differ (P < 0.05).
1
Prepartum cows at 250 d of gestation were supplemented with 1 or 3 mg of cholecalciferol (CH) or calcidiol (CA). Blood was sampled and
analyzed on d 3 and 6 postpartum.
2
Source = effect of source of vitamin D (CH vs. CA); amount = effect of amount of vitamin D (1 vs. 3 mg); source × amount = interaction
between source and amount (CH1 + CA3 vs. CH3 + CA1).
3
Identified by flow cytometry according to size and granularity.
4
Percentage of granulocyte, monocytes, B lymphocytes, or CD14− CD21− cells that were positive for CD62L.
5
MFI = mean fluorescence intensity.
6
Identified by flow cytometry based on expression of cell surface markers.

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 Vieira-Neto et al.: SOURCE AND AMOUNT OF VITAMIN D AND IMMUNE CELLS

Figure 3. Proportion of granulocytes with phagocytosis (A) or phagocytosis with oxidative burst (B), and mean fluorescence intensity (MFI)
for phagocytosis (C) or oxidative burst (D). Prepartum cows were supplemented with 1 or 3 mg of cholecalciferol (CH1 or CH3) or calcidiol (CA1
or CA3) from 250 d of gestation to calving. Error bars depict SEM. Panel A, prepartum: effect of source (P = 0.51), amount (P = 0.06), and
interaction between source and amount (P = 0.93); postpartum: effect of source (P = 0.96), amount (P = 0.42), and interaction between source
and amount (P = 0.84). Panel B, prepartum: effect of source (P = 0.88), amount (P = 0.26), and interaction between source and amount (P =
0.23); postpartum: effect of source (P = 0.47), amount (P = 0.82), and interaction between source and amount (P = 0.35). Panel C, prepartum:
effect of source (P = 0.61), amount (P = 0.06), and interaction between source and amount (P = 0.79); postpartum: effect of source (P = 0.29),
amount (P = 0.37), and interaction between source and amount (P = 0.85). Panel D, prepartum: effect of source (P = 0.42), amount (P = 0.82),
and interaction between source and amount (P = 0.05); postpartum: effect of source (P = 0.67), amount (P = 0.73), and interaction between
source and amount (P = 0.22).

gene involved in Ca binding, CALM3, compared with
cows fed CH (Table 6). Feeding 3mg upregulated (P <
0.05) the expression of DEFB5 but downregulated (P
< 0.05) the expressions of MPO and PP3CA compared
with cows fed 1mg. Interactions (P < 0.05) between
source and amount of vitamin D were observed for LTF,
MMP9, SOD1, and STIM1. The expression of LTF was
greatest in CA1, MMP9 was greatest in CH3, SOD1
was greatest in CH1, and STIM1 was greatest in CA1.
Treatment did not affect the expression of CD44,
ICAM1, ITGAL, ITGAM, ITGB1, ITGB2, LGALS8,
NOD1, NOD2, TLR1, TLR2, TLR4, TLR6, TLR9,
AKT1, FOS, IRAK4, JUN, MAPK1, MAPK3,
MYD88, NFATC1, NFKB1, NFKB2, CCL5, CXCL8,
Journal of Dairy Science Vol. 104 No. 10, 2021
IFNG, IL1B, IL1R2, IL6, IL10, IL23A, TNF, BPI,
CATHL5, CATHL6, CTSB, DEFB1, DEFB10, DEF-
B4A, DEFB6, DEFB7, ELANE, LAP, LYZ, PRKCB,
and TAP, CYBA, CYBB, GPX1, GSR, NCF1, NCF2,
NCF4, ATP2B1, CALM1, CALM2, ITPR1, ORAI1,
PPP3CB, SLC8A1, TRPV5, CYP27B1, RXRA, and
VDR in blood leukocytes isolated from cows in early
postpartum.
DISCUSSION
Cows maintained blood concentrations of vitamin D3
and 25-hydroxyvitamin D3 well above those considered
adequate (Nelson et al., 2016); thus, the treatments
 Vieira-Neto et al.: SOURCE AND AMOUNT OF VITAMIN D AND IMMUNE CELLS

Figure 4. Effect of supplementation of 1 or 3 mg of cholecalciferol (CH1 or CH3) or calcidiol (CA1 or CA3) from 250 d of gestation to calving
on peripheral blood leukocyte gene expression at 270 d of gestation (A) or on d 3 postpartum (B). Heat maps were generated including differ-
ently expressed genes (P < 0.05). Genes separated according to function. Expression increases from red to green. Data based on fold-change of
each gene relative to CH1.

imposed evaluated the effects of manipulating source
and amount of vitamin D in cows with adequate vita-
min D status. As anticipated, source of vitamin D, CH
or CA, had marked influence on concentrations of the
respective vitamins in plasma, and the effect was more
pronounced in those fed 3 versus 1 mg/d. Feeding a
daily dose of 3mg of either source increased the propor-
tions of granulocytes and monocytes pre- and postpar-
tum and, consequently, reduced those of cells of the
adaptive immune system, such as CD14− CD21− cells.
However, cows fed 3mg tended to have reduced per-
centage of granulocytes with phagocytic activity and
reduced MFI for phagocytosis prepartum. Feeding CA
resulted in upregulation of several genes related to cell
adhesion and migration, pathogen recognition receptor,
cell signaling, synthesis of cytokines, and antimicrobial
mechanisms in leukocytes prepartum, with some car-
ryover to the postpartum. In contrast, measures of cell
function investigated were not affect by treatment.
Based on mRNA profiles, it is possible that immune
cells from cows fed CA were better equipped to elicit
a response and potentially to recognize and control
pathogens. Cows fed 3mg CA had greater milk yield
(Poindexter et al., 2019) with no reductions in disease
Journal of Dairy Science Vol. 104 No. 10, 2021
observed; hence, it is possible that CA improved the
immune cell response, mitigating detrimental effects of
unnecessary inflammatory responses observed in severe
cases of disease postpartum, by reducing either the
intensity or the length of the inflammatory response,
but benefits were insufficient to reduce the incidence of
clinical disease.
Cell surface adhesion proteins are essential for
leukocyte migration to peripheral lymph nodes and
into target tissues. Regulation of CD62L controls the
movement of lymphocytes, and once lymphocytes are
activated by antigen-presenting cells in a lymph node,
they shed CD62L from the membrane and move into
blood (Yang et al., 2011). In the current experiment,
we observed an increase in CD62L expression in CD14−
CD21− cells in cows supplemented with 3mg compared
with 1mg, suggesting greater presence of naïve lympho-
cytes in those cows. Manipulations of diet can affect
leukocyte function, as supplemental fatty acids affect
the abundance of granulocytes expressing CD62L,
as well as altering the function of granulocytes from
transition cows (Silvestre et al., 2011). Feeding CH in
the present experiment increased prepartum expres-
sion of CD62L and the proportion of positive cells for
 Vieira-Neto et al.: SOURCE AND AMOUNT OF VITAMIN D AND IMMUNE CELLS

Table 5. Effect of source and amount of vitamin D on relative mRNA expression in leukocytes from prepartum cows for genes affected by
treatment1

CH CA P-value2

Item 1 mg 3 mg   1 mg 3 mg   Source Amount Source × amount

Cell adhesion/migration                  
 CD44 1.00 0.82   1.20 1.28   0.03 0.64 0.33
 ICAM1 1.00 1.56   1.93 1.78   0.05 0.37 0.19
 ITGAL 1.00 0.79   1.16 1.28   0.02 0.59 0.19
 ITGB1 1.00 1.00   1.21 1.33   <0.01 0.58 0.54
 LGALS8 1.00 1.00   1.05 1.22   0.04 0.21 0.20
 SELL 1.00 0.87   1.24 1.29   0.03 0.72 0.54
Pathogen recognition receptor              
 NOD1 1.00 0.84   0.69 0.71   0.01 0.47 0.32
 NOD2 1.00 0.78   1.27 1.29   0.02 0.49 0.43
 TLR2 1.00 0.82   1.12 1.14   <0.01 0.29 0.19
 TLR6 1.00a 0.75b   1.01a 1.18a   0.01 0.48 0.02
Cell signaling                  
 FOS 1.00 1.00   1.56 1.76   <0.01 0.73 0.75
 JUN 1.00 0.96   1.39 1.46   <0.01 0.88 0.65
 NFKB2 1.00 1.14   1.32 1.23   0.02 0.69 0.16
Cytokines                  
 IL1B 1.00 1.20   1.73 1.73   0.05 0.69 0.69
 IL1R1 1.00 0.88   1.39 1.43   0.02 0.79 0.65
 IL1RN 1.00 0.91   1.55 1.46   0.01 0.67 0.91
Antimicrobial mechanisms              
 CTSB 1.00 0.89   1.22 1.37   0.02 1.00 0.38
 LYZ 1.00 0.87   1.41 1.48   <0.01 0.74 0.53
Calcium metabolism                  
 ATP2B1 1.00 0.84   1.02 1.10   0.04 0.48 0.08
 STIM1 1.00 0.94   1.06 1.03   0.05 0.26 0.66
 TRPV5 1.00 0.74   1.51 0.88   0.08 0.01 0.47
a,b
Values within a row with different superscripts differ (P < 0.05).
1
Prepartum cows at 250 d of gestation were supplemented with 1 or 3 mg of cholecalciferol (CH) or calcidiol (CA). Blood was sampled and
mRNA in leukocytes was analyzed on d 270 of gestation. Results are depicted as fold-change relative to cholecalciferol 1 mg.
2
Source = effect of source of vitamin D (CH vs. CA); amount = effect of amount of vitamin D (1 vs. 3 mg); source × amount = interaction
between source and amount (CH1 + CA3 vs. CH3 + CA1).

CD62L in granulocytes of cows, although the effect was
independent of amount of vitamin D fed. In contrast,
Poindexter et al. (2020) observed that supplementation
with 3 mg/d of CA increased CD62L expression in milk
macrophages and granulocytes, although the response
observed was specific to CA compared with 1 mg/d
of CH. The reduced percentage of monocytes CD62L+
and the reduced intensity of CD62L expression in
monocytes in cows fed CA suggest more activation of
those cells, perhaps linked with the observed changes in
mRNA expression with indications of increased abun-
dance of genes involved in recognizing and controlling
pathogens.
Expression of leukocyte mRNA for several cell mi-
gration– and adhesion molecule–related genes were
upregulated by supplementing CA. Of the 11 genes
evaluated related to cell adhesion and migration, 7
were upregulated by CA prepartum, whereas only 3
were upregulated postpartum. Leukocyte migration
is mediated by proteins transcribed by the selectin
genes SELL and SELE, which reduce the rolling veloc-
ity of cells in the endothelium (Bargatze et al., 1994).
This first adherence of leukocytes to endothelial cells
uses CD44 for cell-to-cell interaction and, once cells
are firmly attached, enables signaling and activation
of integrins such as ITGAL and ITGB1. Intracel-
lular integrin domains are interconnected with the
cytoskeleton, and this binding of integrins to actin is
mediated by TLN1 (Calderwood et al., 1999). In fact,
pathogen recognition receptors can also regulate cell
adhesion and migration. Toll-like receptors, such as
TLR2, rapidly activate integrin-dependent leukocyte
adhesion to ICAM1 (Chung et al., 2014). Altogether,
CA supplementation upregulated genes involved in the
pathway for leukocyte recruitment, suggesting that
those leukocytes would potentially be more readily able
to migrate into the target tissue to mount a response
to pathogens. This may be one of the reasons Martinez
et al. (2018) observed reduced retained placenta and
metritis in cows supplemented with CA and Poindexter
et al. (2020) observed increased mRNA expression of
IL1B and inducible nitric oxide synthase in milk so-
matic cells after an intramammary bacterial challenge
in cows supplemented with CA.

Journal of Dairy Science Vol. 104 No. 10, 2021

 Vieira-Neto et al.: SOURCE AND AMOUNT OF VITAMIN D AND IMMUNE CELLS

Defense against bacterial infection relies on recog-
nition of antigens by pathogen recognition receptors.
When activated, membrane receptors such as TLR2
and TLR6 upregulate the genes for COX-2 and PLA2
and the proinflammatory cytokines TNF and IL1b in
a process mediated by MYD88 and NFKB1/2 (Kawai
and Akira, 2007). In addition, IL1B can further expand
the response of the immune system by binding to its re-
ceptor, IL1R1, which can further stimulate the MYD88
and NFKB1/2 cascade, resulting in more proinflamma-
tory elements and amplification of the immune response
(Weber et al., 2010). Additionally, TLR2 can stimulate
the MAPK pathway and activate FOS and JUN, con-
sequently promoting transcription of target genes with
AP-1 binding sites, including several cytokines, such as
IL6, IL10, TNF, and GM-CSF. Calcidiol treatment up-
regulated several genes associated with these pathways
during the prepartum period, including IL1RN, which
is an antagonist of IL1R1 and prevents overstimulation
of the IL1B response (Arend and Guthridge, 2000).
Therefore, it appears that CA induced upregulated
genes with potential for a proinflammatory response,
yet at the same time CA also upregulated IL1RN as
a control check point to prevent potential undesirable
inflammatory responses.
Feeding 3mg tended to reduce granulocyte phago-
cytosis prepartum compared with feeding 1mg treat-
ments, whereas no changes were observed postpartum.
In contrast with our data, Martinez et al. (2018) found
increased percentage of granulocytes positive for oxi-
dative burst postpartum in cows supplemented with
CA. Under bacterial exposure, reactive oxygen species
are produced from superoxide anions and are used for
microbial killing (Paape et al., 2003). A key enzyme
in the synthesis of reactive oxygen species is NADPH
oxidase, a complex of proteins formed by NCF1, NCF2,
and NCF4, which were not affected by vitamin D
supplementation in the present experiment. Thus, the
potential benefits of CA supplementation on phagocy-
tosis and oxidative burst remained unclear based on
the results of this experiment. Nevertheless, enzymes
that can aid during bacterial infection, such as those
conferred by the gene LYZ, were upregulated by CA
during the prepartum period, along with CTSB, the
gene that encodes cathepsin B, an enzyme involved in
supporting phagocytosis and metalloproteinases.
Calcium is an important intracellular second messen-
ger, and activation of cells results in release of endog-
enous ionized Ca and increased uptake from the extra-
cellular space in a process denominated store-operated

Table 6. Effect of source and amount of vitamin D on relative mRNA expression in leukocytes of postpartum cows for genes affected by
treatment1

CH CA P-value2

Item 1 mg 3 mg   1 mg 3 mg   Source Amount Source × amount

Cell adhesion/migration                  
 CXCR2 1.00 0.99   1.44 1.69   0.03 0.69 0.66
 SELL 1.00 1.03   1.39 1.40   0.03 0.88 0.94
 TLN1 1.00 1.04   1.11 1.15   0.03 0.96 0.52
Cell signaling                  
 AKT2 1.00 0.88   0.83 0.80   0.03 0.21 0.44
Cytokines                  
 CCL2 1.00 2.00   3.00 3.49   0.03 0.27 0.47
 IL1R1 1.00 1.18   1.64 1.45   0.04 0.91 0.40
 IL1RN 1.00 1.12   1.55 1.67   0.02 0.60 0.91
Antimicrobial mechanisms                
 DEFB3 1.00 1.33   1.67 1.52   0.05 0.55 0.24
 DEFB5 1.00 2.12   1.44 1.61   0.83 0.05 0.14
 LTF 1.00ab 1.43ab   1.57a 0.65b   0.60 0.41 0.05
 MMP9 1.00b 2.47a   2.18ab 1.40ab   0.73 0.47 0.04
Oxidative burst                  
 MPO 1.00 0.59   1.52 0.79   0.09 <0.01 0.76
 RAC2 1.00 0.95   1.08 1.04   0.03 0.24 0.83
 SOD1 1.00a 0.95ab   0.85b 0.97a   0.12 0.35 0.04
Calcium metabolism                  
 CALM3 1.00 0.92   0.87 0.85   0.01 0.15 0.39
 PP3CA 1.00 0.84   0.91 0.89   0.77 0.03 0.09
 STIM1 1.00ab 0.93b   0.95ab 1.01a   0.57 0.84 0.04
a,b
Values within a row with different superscripts differ (P < 0.05).
1
Prepartum cows at 250 d of gestation were supplemented with 1 or 3 mg of cholecalciferol (CH) or calcidiol (CA). Blood was sampled and
mRNA was quantified in leukocytes analyzed on d 3 postpartum. Results are depicted as fold-change relative to cholecalciferol 1 mg.
2
Source = effect of source of vitamin D (CH vs. CA); amount = effect of amount of vitamin D (1 vs. 3 mg); source × amount = interaction
between source and amount (CH1 + CA3 vs. CH3 + CA1).

Journal of Dairy Science Vol. 104 No. 10, 2021

 Vieira-Neto et al.: SOURCE AND AMOUNT OF VITAMIN D AND IMMUNE CELLS
Ca entry (Bréchard and Tschirhart, 2008). An essential
protein in this process is STIM1, which regulates and
activates the store-operated Ca entry, together with
ORAI1. Reduced concentrations of cytosolic ionized Ca
have been associated with impaired granulocyte phago-
cytosis and oxidative burst in dairy cows (Martinez
et al., 2014). However, vitamin D metabolites are key
regulators of Ca homeostasis, and treatments resulted
in minor changes in expression of genes involved in
intracellular Ca trafficking in leukocytes. Incidence of
milk fever and serum concentrations of total Ca did
not differ among treatments. Perhaps the lack of ef-
fects of treatment on serum concentrations of Ca and
other minerals abolished any potential effect of source
or amount of vitamin D on genes involved in Ca regula-
tory mechanisms in leukocytes.
Evidence exists for the role of vitamin D metabo-
lites on immunity. Intramammary treatment with CA
has been shown to reduce severity of bacterial-induced
mastitis in dairy cows (Lippolis et al., 2011). Merriman
et al. (2018) showed that intramammary treatment
with CA increased abundance of the 1α-hydroxylase
gene CYP27B1 in milk cells, suggesting that CA might
stimulate local conversion of 25-dihydroxyvitamin
D3 into 1α,25-dihydroxyvitamin D3. Their work also
showed that mammary cell inflammation induced by
LPS activates the vitamin D system with upregula-
tion of CYP27B1 and CYP24A1 in cells isolated
from milk. Increased machinery for local synthesis of
1α,25-dihydroxyvitamin D3 by immune cells has been
observed in vitro in human macrophages and in vivo in
bovine monocytes (Liu et al., 2006; Nelson et al., 2010).
Therefore, it is possible that dietary CA provided more
substrate for the synthesis of 1α,25-dihydroxyvitamin
D3 by immune cells (Nelson et al., 2010). In support
of that, increasing CA resulted in greater β-defensin
response (DEFB3, DEFB4, DEFB7, and DEFB10) in
a dose-dependent manner in monocytes from lactating
cows stimulated with LPS in vitro (Merriman et al.,
2015). Thus, vitamin D status might contribute to the
ability of the immune response to confer resistance to
bacterial infections. Although not conclusive, the pres-
ent results suggest that immune cells from cows supple-
mented with CA experience upregulation of genes that
might better equip them to harness an immune response,
thereby providing an opportunity to improve the de-
fense mechanisms in response to peripartum bacterial
diseases. It is noteworthy that feeding CH1 resulted in
a mean concentration of 25-hydroxyvitamin D3 of 57
ng/mL, sufficient to maintain adequate vitamin D sta-
tus (Nelson et al., 2016). The changes observed in this
experiment suggest that providing vitamin D beyond
the amounts needed to prevent perceived deficiency, in
Journal of Dairy Science Vol. 104 No. 10, 2021
particular with supplemental CA, might play a role in
supporting innate immune defenses in dairy cows.
CONCLUSIONS
Peripheral blood leukocyte mRNA expression is
regulated by supplementation of vitamin D, and cal-
cidiol enhanced expression of numerous genes involved
in a multitude of pathways for cell adhesion and migra-
tion, intracellular signaling, and activation of antimi-
crobial mechanisms to eventually eliminate pathogens.
Although a larger number of genes were affected by
calcidiol prepartum than during the early postpartum
period, genes in the same pathways were differentially
expressed and followed a similar pattern pre- and
postpartum, suggesting some carryover effect as lac-
tation starts. Among the genes differently expressed,
those related to cell adhesion and migration, pathogen
recognition receptors, cell signaling, and synthesis of
cytokines were more susceptible to changes. It is likely
that changes in mRNA expression with calcidiol were
induced by the increased substrate for local synthesis of
1α,25-dihydroxyvitamin D3 in immune cells. Neverthe-
less, it is important to note that, despite the observed
changes in concentrations of vitamin D in plasma and
of mRNA in blood leukocytes, treatments were unable
to affect the risk of clinical diseases in the first 60 DIM.
Supplementing calcidiol is a more effective means of
manipulating the concentrations of 25-hydroxyvitamin
D3 in plasma of dairy cows and provides an opportu-
nity to alter leukocyte machinery to harness immune
responses to fend off infections.
ACKNOWLEDGMENTS
The authors thank J. Driver (University of Florida,
Gainesville) for technical assistance and K. C. Jeong
(University of Florida) for preparing the labeled Esch-
erichia coli for granulocytes assays. The help of the
staff of the University of Florida Dairy Research Unit
is greatly appreciated. The authors thank Stephane
Etheve (DSM Nutritional Products, Kaiseraugst, Swit-
zerland) for assistance with vitamin D assays in plasma
samples. Partial funding for this project was provided
by the Southeast Milk Check-Off Dairy Research and
Education Projects (Belleview, FL) and DSM Nutri-
tional Products. The authors have not stated any con-
flicts of interest.
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