Alterations in the gut microbiota of Eimeria infected broiler chickens fed diets

supplemented with varying levels of dietary calcium and phosphorus, along

with 25-hydroxycholecalciferol

Janghan Choi ,*,y Jihwan Lee,* and Woo Kyun Kim *,1
*
Department of Poultry Science, University of Georgia, Athens, GA 30602, USA; and yUS National Poultry Research
Center, USDA-ARS, Athens, GA 30605, USA

ABSTRACT The purpose of the study was to investi- avian beta defensin 5 (AvBD5) in the Eimeria chal-
gate the effects of the reduced dietary calcium (Ca) and lenging condition (interaction; P < 0.05). The reduced
phosphorus (P) level and supplementation of 25-hydrox- dietary Ca and P level tended to decrease the relative
ycholecalciferol (25-OHD3) on the expression of vita- mRNA expression of jejunal AvBD9 in the Eimeria chal-
min D receptor (VDR) and antimicrobial peptides and lenging condition (interaction; P = 0.051). The reduced
gut microbiota of broiler chickens with/without Eimeria dietary Ca and P level decreased observed features
challenge. A total of 576 fourteen-day-old broiler chicks (alpha diversity parameter for richness) in the upper
were randomly allocated according to a 2 £ 2 £ 2 facto- jejunal microbiota in the Eimeria challenging condition
rial design with main effects including Eimeria challeng- (interaction; P < 0.05). The supplementation of 25-
ing (125,000 Eimeria acervulina, 25,000 Eimeria OHD3 decreased the relative abundance of the phylum
maxima, and 25,000 Eimeria tenella), dietary Ca and P Bacteroidetes (P < 0.05) and increased the relative
levels (0.84% Ca and 0.42% available P or 0.64% Ca and abundance of the family Ruminococcaceae (P < 0.05) in
0.22% available P), and supplementation of 25-OHD3 the cecal digesta. The supplementation of 25-OHD3
(3,000 IU/kg) of 6 replicates. Three-way ANOVA was decreased the serum endotoxin level in the Eimeria chal-
performed, and the effects of 3 main factors and their lenging condition (interaction; P < 0.05). Therefore, the
interactions were investigated. The reduced dietary Ca reduced dietary Ca and P level modulated the upper
and P level downregulated cathelicidins 3 (CATH3) in jejunal microbiota via modulating the expression of anti-
the upper jejunum in the Eimeria challenging condition microbial peptides, and the supplementation of 25-
(interaction; P < 0.05). The reduced dietary Ca and P OHD3 favorably modulated the cecal microbiota in
level decreased the relative mRNA expression of jejunal broiler chickens with/without Eimeria challenge.
Key words: calcium, phosphorous, 25-hydroxycholecalciferol, gut microbiota, antimicrobial peptide
2024 Poultry Science 103:104223
https://doi.org/10.1016/j.psj.2024.104223

INTRODUCTION absorption of nutrients (Swaggerty et al., 2022). While

the microbiota in the lower gastrointestinal tract (e.g.,
The microbiota in the gastrointestinal tract of broiler ceca) is considered as the major area for microbial activi-
chickens plays important roles in hydrolyzing host-resis- ties with higher density of microbes, the microbiota in
tant nutrients (e.g., fibers, resistant starch, and polysac- the upper gastrointestinal tract (e.g., duodenum and
charides), inhibiting the colonization of pathogenic jejunum) could be also important because it is closely
bacteria, and producing host-benefiting metabolites associated with gut ecosystem and gut inflammation in
such as amino acids and short chain fatty acids (SCFA) the upper gastrointestinal tract, which is the area for
(Apajalahti, 2005). A stable gut microbiota provides nutrient digestion and absorption in broiler chickens (El
protection against the colonization of pathogens and Aidy et al., 2015; Zhang et al., 2022). Therefore, it is
food-borne pathogens and facilitates the digestion and important to maintain or improve the gut microbiota to
augment production efficiency and ensure food safety
Ó 2024 The Authors. Published by Elsevier Inc. on behalf of Poultry issues in broiler production.
Science Association Inc. This is an open access article under the CC Many studies revealed that diverse challenges such as
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/
4.0/). coccidiosis (Choi et al., 2023a), Salmonella infection
Received June 3, 2024. (Choi et al., 2023b), necrotic enteritis (Goo et al., 2023),
Accepted August 11, 2024.
1
and heat stress (Shi et al., 2019) could negatively affect
Corresponding author: [email protected]

1
2 CHOI ET AL.

the gut microbiota in broiler chickens. Diverse nutri- 2022). The bioavailability of 25-OHD3 was higher
tional interventions including probiotics (Wang et al., than cholecalciferol, which indicates that 25-OHD3
2017), prebiotics (Shang et al., 2018; Kumar et al., can improve growth-promoting effects and bone min-
2019), and plant extracts (Choi et al., 2023a; Choi et al., eralization in broiler chickens (Han et al., 2016;
2022a; Choi et al., 2023b) modulated the gut microbiota Oikeh et al., 2019; Chen et al., 2020). Different levels
of broiler chickens. Potentially, these bioactive com- of dietary Ca and P and supplementation of 25-
pounds mainly modulate the microbiota by directly OHD3 could alter the gut microbiota by modulating
exhibiting antimicrobial effects against pathogenic bac- gut immunity and development of broiler chickens.
teria or providing nutrients for commensal microbes. Moreover, coccidiosis, which is induced by the infec-
However, the gut microbiota can be modulated by com- tion of Eimeria spp., can compromise gut ecosystem
ponents of innate and adaptive mucosal immunity such in broiler chickens (Blake et al., 2020; Teng et al.,
as toll like receptors (TLR), mucus, antimicrobial pepti- 2021a,b; Teng et al., 2020). The absorption and
des, and secretory immunoglobulin A in broiler chickens homeostasis of Ca, P, and Vitamin D can be affected
(Choi and Kim, 2022). Kogut (2017) demonstrated that by Eimeria challenging in broiler chickens (Oikeh et
modulation of the gut microbiota by host immunity has al., 2019). Therefore, the purpose of the study was to
a benefit in that it does not induce antimicrobial resis- investigate the effects of the reduced dietary Ca and
tance of microbes. P level and supplementation of 25-OHD3 on the
While the major role of dietary calcium (Ca) and expression of VDR, antimicrobial peptides, and TLR,
phosphorous (P) is bone development and mainte- activities of brush border digestive enzymes, antioxi-
nance, dietary Ca and P are also involved in numer- dant capacity, and microbiota in the upper jejunum
ous biological processes such as cell signaling, cell and ceca of broiler chickens in the general or Eimeria
growth, protein synthesis, etc. (Veum, 2010). Many challenging condition.
studies demonstrated that inadequate levels of die-
tary Ca and P compromised growth performance and
gut health of broiler chickens (Shang et al., 2015;
Xing et al., 2020; Wang et al., 2021; Shi et al., 2023; MATERIALS AND METHODS
Shi et al., 2024), which may suggest that the different Animals and Experimental Design
level of dietary Ca and P can affect the gut micro-
biota in broiler chickens. Because vitamin D regulates The Institutional Animal Care and Use Committee of
the homeostasis of dietary Ca and P, vitamin D is University of Georgia (Athens, GA) approved the ani-
also related to bone development in broiler chickens mal use and procedure of the study (A2021 12-012). A
(Heaney, 2008; Kim et al., 2011). However, a number total of 576 fourteen-day-old male broiler chicks (Cobb
of studies showed that vitamin D and its metabolites 500) were randomly allocated in cages according to a
can influence immune system of broiler chickens 2 £ 2 £ 2 factorial design with Eimeria spp. challenging
mainly by altering the expression of vitamin D recep- (oral gavage of water or oral gavage of 125,000 Eimeria
tor (VDR) (Shojadoost et al., 2015; Zhang et al., acervulina, 25,000 Eimeria maxima, and 25,000 Eimeria
2016). The VDR is known to regulate components of tenella on D 14), dietary Ca and P levels (0.84% Ca and
immune system including macrophages (Shojadoost 0.42% available P or 0.64% Ca and 0.24% available P),
et al., 2015), TLRs (Arababadi et al., 2018), cyto- and supplementation of 25-OHD3 (3,000 IU/kg). Each
kines (Aggeletopoulou et al., 2022), and antimicrobial experimental group had 6 replicates of 12 birds per pen.
peptides (Zhang et al., 2016). 25-hydroxycholecalci- The experimental groups were described in Table 1.
ferol (25-OHD3) formed via hepatic hydroxylation Diets were formulated to meet or exceed the nutrient
from cholecalciferol (vitamin D3) can partially and requirement levels according to Cobb 500 nutritional
fully replace the most common form of vitamin D in guide (Cobb-Vantress, 2018) as shown in the companion
animal feeds (cholecalciferol) (L€ utkeD€orhoff et al., study (Lopes et al., 2024). The vitamin premix, which

Table 1. Description for experimental groups.1

Supplementation of 25-hydroxy
Experimental groups Eimeria challenging Ca (Ca) & phosphorous (P) cholecalciferol (25-OHD3)
Treatment 1 (T1) Oral gavage of PBS 0.84% Ca and 0.44% available P 0 IU/kg
T2 Oral gavage of PBS 0.64% Ca and 0.24% available P 0 IU/kg
T3 Oral gavage of PBS 0.84% Ca and 0.44% available P 3,000 IU/kg
T4 Oral gavage of PBS 0.64% Ca and 0.24% available P 3,000 IU/kg
T5 Oral gavage of 125,000 E. acervulina, 25,000 0.84% Ca and 0.44% available P 0 IU/kg
E. maxima, and 25,000 E. tenella
T6 Oral gavage of 125,000 E. acervulina, 25,000 0.64% Ca and 0.24% available P 0 IU/kg
E. maxima, and 25,000 E. tenella
T7 Oral gavage of 125,000 E. acervulina, 25,000 0.84% Ca and 0.44% available P 3,000 IU/kg
E. maxima, and 25,000 E. tenella
T8 Oral gavage of 125,000 E. acervulina, 25,000 0.64% Ca and 0.24% available P 3,000 IU/kg
E. maxima, and 25,000 E. tenella
 MICROBIOTA WITH DIETATY CA, P, AND 25-OHD3 3
was included in all diets, included 440,917 IU/kg of cho- of each PCR product. Several PCR products from each
lecalciferol (Vitamin D3) gene were stained with 6 £ DNA loading dye (Thermo
Fisher Scientific, Waltham, MA), electrophoresed on a
3% agarose gel in a Tris-acetate-EDTA buffer, and visu-
Sampling alized by adding ethidium bromide to confirm the speci-
On 5 d post inoculation (dpi), one bird per pen was ficity of each PCR product. The geometric mean of Ct
randomly selected and euthanized via cervical disloca- values of glyceraldehyde 3-phosphate dehydrogenase
tion (Teng et al., 2021a). Blood was immediately drawn (GAPDH) and beta-actin were used as reference values
from the heart and collected with heparin-free vacu- to normalize all target genes’ mRNA abundance (Van-
tainers (Grainer Bio-One, Kremsmuenster, Austria). desompele et al., 2002). Relative mRNA abundance of
Collected blood samples were stood for 1 h to allow clot- target genes was determined by using the 2ΔΔCt method
ting and centrifuged at 1,000 £ g for 10 min to recover (Livak and Schmittgen, 2001), and the T1 group (con-
serum. On 6 dpi, one bird per pen was randomly selected trol) was selected as reference group. Each sample was
and euthanized via cervical dislocation. Both tissue and analyzed in duplicate, and the negative control, contain-
digesta samples were collected from the upper jejunum ing water instead of cDNA, was included in each run.
(10 cm below duodenum-jejunum junction) and ceca,
and snap frozen. Collected serum, tissue, and digesta
samples were stored at - 80°C for further analyses. Brush Border Digestive Enzyme Activities,
Total Antioxidant Capacity, and Serum
Endotoxin Level
RNA Extraction and Quantitative Real-Time
Reverse Transcription PCR Approximately 100 mg of upper jejunum and ceca tis-
sues were homogenized in PBS using a beads beater
Approximately 100 mg of upper jejunum and ceca tis- (Biospec Products, Bartlesville, OK). The samples were
sues were homogenized in QIAzol lysis reagents (Qiagen, centrifuged at 12,000 £ g for 15 min at 4°C, and the pro-
Valencia, CA) using a beads beater (Biospec Products, tein concentrations of the supernatants were analyzed
Bartlesville, OK) (Santos et al., 2020). RNA was iso- using Pierce BCA protein assay kits according to the
lated according to the manufacturer’s procedure, and manufacturer’s procedure (Thermo Fisher Scientific,
RNA quantity and quality were measured using a Nano- Waltham, MA). The activities of maltase and sucrase in
Drop 2000 spectrophotometer (Thermo Fisher Scien- the supernatants were analyzed according to the proce-
tific, Waltham, MA). One microgram of RNA was used dure of Lackeyram et al. (2010). Briefly, 100 mL super-
to produce the first-strand cDNA using high-capacity natants were mixed with 400 mL maltose (75 mM) and
cDNA synthesis kits (Applied Biosystems, Foster City, sucrose solution (75 mM), separately and incubated at
CA). Primers used in the study are shown in Table 2. 41°C for 30 min. Afterwards, the concentrations of glu-
Quantitative real-time reverse transcription PCR cose were determined using a Glucose Oxidase Reagent
(qRT-PCR) was performed using SYBR Green Master Set (Pointe Scientific, Canton, MI) according to the
Mix with a Step One thermocycler (Applied Biosystems, manufacturer’s procedure. The activities of lipase in the
Foster City, CA). The final PCR volume (10 mL) was 5 supernatants was determined according to the method
mL of SYBR Green Master Mix, 1.5 mL of cDNA, 0.5 mL of Elgharbawy et al. (2018), the 10-time diluted super-
of forward and reverse primers (10 mM), and 2.5 mL of natants (60 mL) were incubated with 1 mg/mL p-nitro-
water. Thermal cycle conditions for all reactions were as phenyl palmitate solution (Sigma-Aldrich Co., St Louis,
follows: 95°C denature for 10 min, 40 cycles at 95°C for MO; 140 mL) at 41°C for 30 min. The activities of leucine
15 s and 60°C for 1 min, 95°C for 15 s, 60°C for 1 min aminopeptidase (LAP) were assayed by incubating 100
and 95°C for 15 s (Castro et al., 2020). The melting mL supernatant with 100 mL 1 mg/mL l-leucine-p-nitro-
curve of each gene was checked to confirm the specificity anilide solution (Sigma-Aldrich Co., St Louis, MO) at

Table 2. Primers used in the study.1

Forward (50 to 30 ) Reverse (50 to 30 ) Amplicon Accession number

VDR ATGTTCACCTGTCCGTTCAA TTCATCATCCCAATGTCCAC 104 NM_205098.2
AvBD3 CCACCCAGTGCAGAATAAGA GCTCTTCCACAGCAGGAAAT 106 NM_204650.2
AvBD5 CTCTTTGCTGTCCTCCTCCT CTGGAGGACATGACTTGTGG 118 NC_052534.1
AvBD9 GCTGACACCTTAGCATGCAG CATTTGCAGCATTTCAGCTT 113 KR136304.1
CATH3 GCTGTGGACTCCTACAACCA CCATGATGGTGAAGTTGAGG 124 NM_001311177.2
LEAP2 TATTCTTCTCGCTGCTGCTC AGGCTCCAACAGGTCTCAGT 123 NM_001001606.2
TLR2 CGGTGGAAAGGGAGAAAG CTTGCCACATCAGCTTCATT 103 NM_204278.1
TLR3 GGCTAAACGACACTCAAGCA CTTGCAGGCTGAGGTATCAA 113 NC_052535.1
TLR4 CCTGGACTTGGACCTCAGTT TTGTATGGATGTGGCACCTT 110 AY064697.1
TLR5 CGTTAGTGAGAATGGCTGGA TGAGCCCATTGTATGAGAGC 106 XM_040697311.1
GAPDH GCTAAGGCTGTGGGGAAAGT TCAGCAGCAGCCTTCACTAC 161 NM_204305.2
Beta actin CAACACAGTGCTGTCTGGTGGTA ATCGTACTCCTGCTTGCTGATCC 205 NM_205518.2
1
VDR: vitamin D receptor; AvBD: avian beta defensin; CATH3: Cathelicidin 3; LEAP2: liver-expressed antimicrobial peptide 2; TLR: toll like recep-
tor; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.
4 CHOI ET AL.

41°C for 30 min according to Maroux et al. (1973). To between means were compared using the Tukey’s HSD
determine activities of alkaline phosphatase in the tissue (honestly significant difference) test. Statistical signifi-
or in the serum, the 20 ml tissue supernatant (2-time cance was set at P < 0.05, and trends (0.05 ≤ P ≤ 0.1)
dilution) and serum (10-time dilution) were incubated were also presented. Selected parameters with statistical
with 180 mL 10 mM p-nitrophenyl phosphate solution at differences (P < 0.10) in interactions are shown in
41°C for 60 min according to Lackeyram et al. (2010). figures.
The absorbance of the end products (p-nitrophenyl and
p-nitroanilide) was determined at 400 nm by using a
spectrophotometer (VICTOR Nivo, Perkin Elmer, Pon-
RESULTS
tyclun, United Kingdom) and quantify using a prepared
standard curve. The activities of the brush border diges- Relative mRNA Expression of VDR,
tive enzymes were expressed as their end-product level Antimicrobial Peptides, and TLR in the
per mg protein per min. The activities of serum alkaline Jejunum and Ceca
phosphatase were expressed as their end-product level
per mL serum per min. Relative mRNA expression of VDR, antimicrobial
Total antioxidant capacity (TAC) of the upper jeju- peptides, and TLR in the jejunum of broiler chickens
num and ceca tissues was measured in the collected infected with Eimeria spp. and fed different levels of Ca,
supernatant by using a commercial kit (QuantiCromAn- P, and 25-OHD3 are shown in Table 3 and Figure 1.
tioxidant Assay Kit, BioAssay Systems, Hayward, CA) While statistical differences were not observed, reduced
after 2-time sample dilution (Tompkins et al., 2023). dietary Ca and P numerically decreased relative mRNA
Serum endotoxin concentrations were determined using expression of jejunal VDR (P = 0.106). The reduced die-
a Pierce LAL Chromogenic Endotoxin Quantitation tary Ca and P level decreased the relative mRNA
Kit (Thermo Fisher Scientific, Waltham, MA) according expression of jejunal avian beta defensin 5 (AvBD5) in
to the manufacturer’s protocol after 10-time sample the Eimeria challenging condition (interaction; P <
dilution. 0.05). The reduced dietary Ca and P level tended to
decrease the relative mRNA expression of jejunal
AvBD9 in the Eimeria challenging condition (interac-
DNA Extraction and 16s rRNA Analysis tion; P = 0.051). Eimeria challenging significantly
increased relative mRNA expression of jejunal cathelici-
DNA was extracted from the contents of mid-ceca by
din 3 (CATH3), whereas the reduced dietary Ca and P
using QIAamp DNA stool mini kits (Qiagen GmbH, Hil-
level tended to decrease relative mRNA expression of
den, Germany) according to manufacturer procedure.
jejunal CATH3 (P = 0.062). The reduced dietary Ca
The quality and quantity of extracted DNA was checked
and P level decreased the relative mRNA expression of
using a NanoDrop 2000spectrophotometer (Thermo
jejunal CATH3 in the Eimeria challenging condition
Fisher Scientific). The 16s rRNA gene sequencing was
(interaction; P < 0.05). Eimeria challenging significantly
conducted by LC sciences, LLC (Houston, TX) as
decreased relative mRNA expression of jejunal liver-
described by Choi et al. (2022b), QIIME2 (version
expressed antimicrobial peptide 2 (LEAP2). The
2021.11) was used (Bolyen et al., 2019) for the 16s
reduced dietary Ca and P level tended to decrease the
rRNA analysis. By using the QIIME2 demux emp-paired
relative mRNA expression of jejunal TLR3 in the Eime-
function and QIIME2 plugin DADA2, sequences were
ria challenging condition (interaction; P = 0.067). The
demultiplexed and denoised, respectively. A phyloge-
reduced dietary Ca and P level decreased the relative
netic tree of ASV was created, and the taxonomy of
mRNA expression of jejunal TLR5 in the Eimeria chal-
each ASV was classified by using a pretrained classifier
lenging condition (interaction; P < 0.05).
the reference SILVA database, and the phylum- and
Relative mRNA expression of VDR, antimicrobial
family-level composition was presented (Yilmaz et al.,
peptides, and TLRs in the ceca of broiler chickens
2014). Alpha diversity was analyzed by QIIME2’s built-
infected with Eimeria spp. and fed different levels of
in functions.
Ca, P, and 25-OHD3 is presented in Table 4. Eime-
ria challenging significantly upregulated mRNA
Statistical Analysis expression of cecal VDR. Eimeria challenging signifi-
cantly upregulated mRNA expression of cecal
SAS (version 9.4; SAS Inst. Inc., Cary, NC) and AvBD3, AvBD9, and CATH3. The relative mRNA
GraphPad Prism (Version 9.1.0; GraphPad Software, expression of LEAP2 tended to be decreased by
San Diego, CA) were used for statistical analyses and Eimeria challenging (P = 0.068). The relative
graph construction. All groups were compared using mRNA expression of TLR3 was significantly downre-
PROC MIXED based on a 2 £ 2 £ 2 factorial design gulated by Eimeria challenging. The relative mRNA
with main effects of Eimeria challenging (E), dietary Ca expression of TLR4 and TLR5 was significantly
and P level (Ca&P), and 25-OHD3 supplementation upregulated and downregulated, respectively, by
(25-OHD3). The interactions (E £ Ca&P, E £ 25- Eimeria challenging. There were interactions
OHD3, Ca&P £ 25-OHD3, and E £ Ca&P £ 25-OHD3) between the dietary Ca and P level and supplemen-
among the main effects were investigated. Differences tation of 25-OHD3 in the Eimeria challenging
 MICROBIOTA WITH DIETATY CA, P, AND 25-OHD3 5
Table 3. Effects of the reduced dietary calcium (Ca) and phosphorous (P) level with the supplementation of 25-hydroxycholecalciferol
(25-OHD3) on the relative mRNA abundance of vitamin D receptor, antimicrobial peptides, and toll like receptors in the upper jejunum
of broiler chickens in the general and Eimeria challenging condition.1
E Ca&P 25-OHD3 VDR AvBD3 AvBD5 AvBD9 CATH3 LEAP2 TLR2 TLR3 TLR4 TLR5

T1 NC N NS 1.308 1.934 1.724 1.529 1.559b 1.529 1.187 1.458 1.379 1.186
T2 NC R NS 0.959 4.244 2.571 9.767 1.519b 1.222 1.595 1.176 1.602 1.863
T3 NC N S 1.325 1.858 1.778 2.462 1.228b 1.450 3.310 1.548 2.357 1.997
T4 NC R S 1.429 2.947 2.602 4.951 1.500b 1.774 3.212 2.136 1.819 2.545
T5 C N NS 1.283 13.02 3.173 15.85 4.400ab 0.456 4.492 3.477 2.293 1.933
T6 C R NS 0.477 2.412 1.927 4.906 3.015ab 0.076 2.469 1.354 1.979 1.118
T7 C N S 1.267 11.74 3.827 14.14 5.896a 0.520 5.734 3.303 2.785 2.117
T8 C R S 0.419 2.381 1.598 3.391 2.624ab 0.141 2.064 1.403 1.616 0.925
SEM2 0.994 12.29 2.021 0.994 1.994 1.107 3.182 1.992 1.751 1.327
P value
Treatments3 0.464 0.564 0.497 0.464 0.002 0.054 0.242 0.293 0.875 0.383
E 0.178 0.198 0.433 0.231 <0.001 0.001 0.146 0.169 0.458 0.334
Ca&P 0.106 0.250 0.445 0.500 0.062 0.564 0.151 0.114 0.379 0.613
25-OHD3 0.720 0.851 0.861 0.661 0.744 0.640 0.220 0.689 0.517 0.339
E £ Ca&P 0.227 0.107 0.033 0.051 0.040 0.547 0.110 0.067 0.567 0.041
E £ 25-OHD3 0.628 0.996 0.919 0.968 0.531 0.789 0.434 0.612 0.600 0.333
Ca&P £ 25-OHD3 0.721 0.999 0.669 0.732 0.498 0.624 0.561 0.637 0.429 0.742
E £ Ca&P £ 25-OHD3 0.668 0.863 0.683 0.714 0.345 0.626 0.758 0.780 0.963 0.873
1
E: Eimeria challenging (oral gavage of water or oral gavage of 125,000 Eimeria acervulina, 25,000 Eimeria maxima, and 25,000 Eimeria tenella on D
14), NC: Eimeria non-challenged, C: Eimeria challenged, Ca&P: dietary calcium and phosphorus levels (0.84% Ca and 0.42% available P or 0.64% Ca
and 0.24% available P), N: normal dietary Ca and P level, R: reduced dietary Ca and P level, 25-OHD3: supplementation of 25-OHD3 (3,000 IU/kg), NS:
25-OHD3 non-supplemented, S: 25-OHD3 supplemented, T: treatment, VDR, vitamin D receptor; AvBD, avian beta defensin; CATH3, Cathelicidin 3;
LEAP2, liver-expressed antimicrobial peptide 2; TLR, toll like receptor. All groups were compared using PROC MIXED based on a 2 £ 2 £ 2 factorial
design with main effects of Eimeria challenging (E), dietary Ca and P level (Ca&P), and 25-OHD3 supplementation (25-OHD3). The interactions
(E £ Ca&P, E £ 25-OHD3, Ca&P £ 25-OHD3, and E £ Ca&P £ 25-OHD3) among the main effects were investigated. Different letters within the same
column indicates significant differences according to the Tukey’s HSD (honestly significant difference) test (P < 0.05).
2
SEM: Standard error of the mean.
3
P values for all treatments comparison.

Figure 1. Interactions between Eimeria challenging (E), the dietary calcium (Ca) and phosphorous (P) levels (Ca&P), and the supplementa-
tion of 25-hydroxycholecalciferol (25-OHD3) on the relative mRNA abundance of antimicrobial peptides (AvBD5, AvBD9, and CaTH3) and toll
like receptors (TLR3 and TLR5) in the upper jejunum and ceca of broiler chickens. Selected parameters with statistical differences (P < 0.10) in
interactions are shown. Different letters indicate statistically significant differences (P < 0.05). NC: Eimeria non-challenged; C: Eimeria challenged;
N: normal dietary Ca and P level; N: normal dietary Ca and P level; R: reduced dietary Ca and P level; NS: 25-OHD3 non-supplemented; S: 25-
OHD3 supplemented; AvBD: avian beta defensin; CATH3, Cathelicidin 3; TLR: toll like receptors
6 CHOI ET AL.

Table 4. Effects of the reduced dietary calcium (Ca) and phosphorous (P) level with the supplementation of 25-hydroxycholecalciferol
(25-OHD3) on the relative mRNA abundance of vitamin D receptor, antimicrobial peptides, and toll like receptors in the ceca of broiler
chickens in the general and Eimeria challenging condition.1

E Ca&P 25-OHD3 VDR AvBD3 AvBD5 AvBD9 CATH3 LEAP2 TLR2 TLR3 TLR4 TLR5
T1 NC N N 1.240 1.084 1.127 1.138 1.269 1.043 1.090 1.100 1.073bc 1.161ab
T2 NC L N 1.233 1.131 2.044 1.196 1.320 1.637 0.788 1.412 0.802c 2.669a
T3 NC N S 1.265 1.067 0.978 1.405 2.075 1.515 1.110 1.387 0.937bc 1.307ab
T4 NC L S 1.243 0.837 0.863 1.077 1.427 1.883 1.063 1.448 1.276bc 1.006b
T5 C N N 3.669 4.131 0.525 4.416 5.226 0.889 1.014 1.100 1.931ab 0.668b
T6 C L N 4.505 3.894 1.596 3.711 4.322 1.309 1.011 0.650 2.432a 0.327b
T7 C N S 2.799 2.420 0.834 3.483 5.937 1.003 1.548 0.763 1.757abc 0.614b
T8 C L S 3.738 3.461 1.408 1.138 3.491 0.877 1.024 0.871 1.555abc 0.411b
SEM2 2.921 1.485 2.077 3.699 0.925 0.508 0.639 0.594 0.892
P value
Treatments3 0.024 0.240 0.715 0.240 0.182 0.448 0.408 0.261 <0.001 0.002
E <0.001 0.006 0.708 0.002 0.004 0.068 0.357 0.012 <0.001 <0.001
Ca&P 0.471 0.855 0.162 0.671 0.361 0.246 0.143 0.968 0.596 0.528
25-OHD3 0.508 0.471 0.484 0.661 0.854 0.709 0.159 0.782 0.306 0.162
E £ Ca&P 0.456 0.771 0.625 0.808 0.523 0.536 0.763 0.344 0.737 0.101
E £ 25-OHD3 0.490 0.590 0.403 0.591 0.810 0.337 0.670 0.560 0.050 0.146
Ca&P £ 25-OHD3 0.971 0.769 0.378 0.991 0.603 0.474 0.653 0.683 0.892 0.117
E £ Ca&P £ 25-OHD3 0.962 0.647 0.757 0.786 0.845 0.766 0.192 0.285 0.063 0.069
1
E: Eimeria challenging (oral gavage of water or oral gavage of 125,000 Eimeria acervulina, 25,000 Eimeria maxima, and 25,000 Eimeria tenella on D
14), NC: Eimeria non-challenged, C: Eimeria challenged, Ca&P: dietary calcium and phosphorus levels (0.84% Ca and 0.42% available P or 0.64% Ca
and 0.24% available P), N: normal dietary Ca and P level, R: reduced dietary Ca and P level, 25-OHD3: supplementation of 25-OHD3 (3,000 IU/kg), NS:
25-OHD3 non-supplemented, S: 25-OHD3 supplemented, T: treatment, VDR, vitamin D receptor; AvBD, avian beta defensin; CATH3, Cathelicidin 3;
LEAP2, liver-expressed antimicrobial peptide 2; TLR, toll like receptor. All groups were compared using PROC MIXED based on a 2 £ 2 £ 2 factorial
design with main effects of Eimeria challenging (E), dietary Ca and P level (Ca&P), and 25-OHD3 supplementation (25-OHD3). The interactions
(E £ Ca&P, E £ 25-OHD3, Ca&P £ 25-OHD3, and E £ Ca&P £ 25-OHD3) among the main effects were investigated. Different letters within the same
column indicates significant differences according to the Tukey’s HSD (honestly significant difference) test (P < 0.05).
2
SEM: Standard error of the mean.
3
P values for all treatments comparison.

condition on cecal TLR4 (tendency; P = 0.063) and shown in Table 6. Eimeria challenging significantly
in cecal TLR5 (tendency; P = 0.069). decreased the activities of serum alkaline phosphatase
and significantly increased serum endotoxin level. How-
ever, 25-OHD3 supplementation significantly decreased
Brush Border Digestive Enzyme Activities serum endotoxin level in the Eimeria challenging condi-
and TAC in the Jejunum and Ceca tion (interaction; P < 0.05).
Brush border digestive enzyme activities and TAC in
the jejunum and ceca of broiler chickens infected with
Eimeria spp. and fed different levels of Ca, P, and 25- Alpha and Beta Diversity of the Microbiota in
OHD3 are shown in Table 5 and Figure 2. Eimeria chal- the Jejunal and Cecal Digesta
lenging significantly decreased activities of jejunal
Alpha diversity of the microbiota in the jejunal and
sucrase, maltase, and IAP and tended to decrease activi-
cecal digesta of broiler chickens infected with Eimeria
ties of jejunal lipase (P = 0.064). The activities of jejunal
spp. and fed different levels of Ca, P, and 25-OHD3 are
sucrase was increased when fed reduced dietary Ca and
shown in Table 7 and Figure 3. Eimeria infection tended
P level with the supplementation of 25-OHD3 (interac-
to decrease faith phylogenetic diversity (phylogenetic
tion; P < 0.05). The activities of jejunal maltase tended
differences; P = 0.081) and significantly decreased
to be reduced in the Eimeria challenging condition
observed features (richness) in the upper jejunal micro-
(interaction; P = 0.060). The supplementation of 25-
biota. Observed features of the microbiota in the jejunal
OHD3 tended to increase the activities of jejunal lipase
digesta was reduced in the Eimeria challenging condi-
(P = 0.096). Eimeria challenging significantly decreased
tion (interaction; P < 0.05). The reduced level of dietary
the activities of cecal IAP. No significant difference was
Ca and P with the supplementation of 25-OHD3 tended
observed in TAC of the jejunum and ceca among the
to decrease pielou’s evenness (P = 0.075) shannon
treatments (P > 0.10).
entropy (richness and evenness; P = 0.082) of the micro-
biota in the jejunal digesta (interaction). In the cecal
Activities of Serum Alkaline Phosphatase digesta, the alpha diversity parameters including pie-
and Serum Endotoxin Level lou’s evenness, faith phylogenetic diversity, observed
features, and shannon entropy were significantly
Activities of serum alkaline phosphatase and serum decreased due to Eimeria infection. However, different
endotoxin level in broiler chickens infected with Eimeria levels of Ca, P, and 25-OHD3 did not modulate the alpha
spp. and fed different levels of Ca, P, and 25-OHD3 are diversity parameters in the cecal digesta (P > 0.10).
 MICROBIOTA WITH DIETATY CA, P, AND 25-OHD3 7
Table 5. Effects of the reduced dietary calcium (Ca) and phosphorous (P) level with the supplementation of 25-hydroxycholecalciferol
(25-OHD3) on the activities of brush border digestive enzymes including sucrase (nmol glucose released/mg protein/min), maltase
(nmol glucose released/mg protein/min), intestinal alkaline phosphatase (IAP; mmol p-nitrophenol liberated/mg protein/min), leucine
aminopeptidase (LAP; mmol p-nitrophenol liberated/mg protein/min), and lipase (mmol p-nitrophenyl phosphate liberated/mg pro-
tein/min) in the upper jejunum and ceca of broiler chickens in the general and Eimeria challenging condition1

Jejunum Ceca
E Ca&P 25-OHD3 Sucrase Maltase IAP LAP Lipase IAP
T1 NC N N 0.218a 0.734ab 0.259abc 31.94 0.473 0.225
T2 NC L N 0.191ab 0.748ab 0.272ab 31.96 0.499 0.207
T3 NC N S 0.129ab 0.629ab 0.241abcd 31.04 0.525 0.204
T4 NC L S 0.227a 0.815a 0.293a 31.50 0.523 0.218
T5 C N N 0.162ab 0.597ab 0.138d 33.98 0.452 0.178
T6 C L N 0.075b 0.452b 0.145cb 32.19 0.478 0.165
T7 C N S 0.141ab 0.570ab 0.134d 32.37 0.476 0.154
T8 C L S 0.163ab 0.552ab 0.164bcd 31.54 0.489 0.172
SEM2 0.077 0.162 0.064 4.67 0.056 0.042
P value
Treatments3 0.033 0.007 <0.001 0.982 0.334 0.042
E 0.016 <0.001 <0.001 0.503 0.064 0.001
Ca&P 0.956 0.843 0.177 0.693 0.343 0.975
25-OHD3 0.889 0.854 0.798 0.505 0.096 0.564
E £ Ca&P 0.139 0.060 0.718 0.569 0.820 0.860
E £ 25-OHD3 0.187 0.553 0.875 0.867 0.534 0.886
Ca&P £ 25-OHD3 0.013 0.119 0.395 0.796 0.554 0.208
E £ Ca&P £ 25-OHD3 0.868 0.814 0.830 0.925 0.817 0.984
1
E: Eimeria challenging (oral gavage of water or oral gavage of 125,000 Eimeria acervulina, 25,000 Eimeria maxima, and 25,000 Eimeria tenella on D
14), NC: Eimeria non-challenged, C: Eimeria challenged, Ca&P: dietary calcium and phosphorus levels (0.84% Ca and 0.42% available P or 0.64% Ca
and 0.24% available P), N: normal dietary Ca and P level, R: reduced dietary Ca and P level, 25-OHD3: supplementation of 25-OHD3 (3,000 IU/kg), NS:
25-OHD3 non-supplemented, S: 25-OHD3 supplemented, T: treatment, IAP: intestinal alkaine phosphatase, LAP, leucine aminopeptidase. All groups
were compared using PROC MIXED based on a 2 £ 2 £ 2 factorial design with main effects of Eimeria challenging (E), dietary Ca and P level (Ca&P),
and 25-OHD3 supplementation (25-OHD3). The interactions (E £ Ca&P, E £ 25-OHD3, Ca&P £ 25-OHD3, and E £ Ca&P £ 25-OHD3) among the
main effects were investigated. Different letters within the same column indicates significant differences according to the Tukey’s HSD (honestly signifi-
cant difference) test (P < 0.05).
2
SEM: Standard error of the mean.
3
P values for all treatments comparison.

Taxa Abundance Proteobacteria, Bacteroidetes, and Actinobacteria

were the most abundant phylum groups in the upper
Relative abundance of microbial composition at the jejunal digesta. Eimeria challenging significantly
phylum level in the upper jejunal digesta of broiler decreased the relative abundance of phylum Firmi-
chickens infected with Eimeria spp. and fed different cutes, Cyanobacteria, and Actinobacteria in the
levels of Ca, P, and 25-OHD3 is presented in Table 8 microbiota of the upper jejunal digesta. In the micro-
and Figure 4. The phylum Firmicutes, Cyanobacteria, biota of the upper jejunal digesta, the relative

Figure 2. Interactions between Eimeria challenging (E), the dietary calcium (Ca) and phosphorous (P) levels (Ca&P), and the supplementa-
tion of 25-hydroxycholecalciferol (25-OHD3) on the activities of brush border digestive enzymes including jejunal sucrase (nmol glucose released/
mg protein/min) and jejunal maltase (nmol glucose released/mg protein/min) and serum endotoxin level (EU/mL) in broiler chickens. Selected
parameters with statistical differences (P < 0.10) in interactions are shown. Different letters indicate statistically significant differences (P < 0.05).
NC: Eimeria non-challenged; C: Eimeria challenged; N: normal dietary Ca and P level; R: reduced dietary Ca and P level; NS: 25-OHD3 non-supple-
mented, S: 25-OHD3 supplemented.
8 CHOI ET AL.

Table 6. Effects of the reduced dietary calcium (Ca) and phosphorous (P) level with the supplementation of 25-hydroxycholecalciferol
(25-OHD3) on the total antioxidant capacity (TAC; mM Trolox Equivalents/mg protein) in the upper jejunum and ceca, activities of
serum alkaline phosphatase (mmol p-nitrophenol liberated/mL serum/min), and serum endotoxin level (EU/mL) in broiler chickens in
the general and Eimeria challenging condition1

TAC
Serum alkaline Serum
E Ca&P 25-OHD3 Jejunum Ceca phosphatase endotoxin
T1 NC N N 49.47 36.00 2.670 0.688b
T2 NC L N 50.71 35.41 3.396 0.665b
T3 NC N S 45.31 34.01 3.214 0.837ab
T4 NC L S 45.29 33.35 3.214 1.190ab
T5 C N N 53.23 35.49 2.891 1.445ab
T6 C L N 47.37 36.87 2.696 1.770a
T7 C N S 48.67 37.00 2.767 1.228ab
T8 C L S 52.32 31.21 2.714 0.805ab
2
SEM 6.074 4.870 0.282 0.578
P value
Treatments3 0.215 0.465 0.242 0.017
E 0.131 0.751 0.045 0.008
Ca&P 0.890 0.321 0.492 0.730
25-OHD3 0.198 0.152 0.712 0.451
E £ Ca&P 0.628 0.576 0.166 0.525
E £ 25-OHD3 0.163 0.987 0.500 0.008
Ca&P £ 25-OHD3 0.247 0.206 0.402 0.581
E £ Ca&P £ 25-OHD3 0.133 0.215 0.216 0.100
1
E: Eimeria challenging (oral gavage of water or oral gavage of 125,000 Eimeria acervulina, 25,000 Eimeria maxima, and 25,000 Eimeria tenella on D
14), NC: Eimeria non-challenged, C: Eimeria challenged, Ca&P: dietary calcium and phosphorus levels (0.84% Ca and 0.42% available P or 0.64% Ca
and 0.24% available P), N: normal dietary Ca and P level, R: reduced dietary Ca and P level, 25-OHD3: supplementation of 25-OHD3 (3,000 IU/kg), NS:
25-OHD3 non-supplemented, S: 25-OHD3 supplemented, T: treatment. All groups were compared using PROC MIXED based on a 2 £ 2 £ 2 factorial
design with main effects of Eimeria challenging (E), dietary Ca and P level (Ca&P), and 25-OHD3 supplementation (25-OHD3). The interactions
(E £ Ca&P, E £ 25-OHD3, Ca&P £ 25-OHD3, and E £ Ca&P £ 25-OHD3) among the main effects were investigated. Different letters within the same
column indicates significant differences according to the Tukey’s HSD (honestly significant difference) test (P < 0.05).
2
SEM: Standard error of the mean.
3
P values for all treatments comparison.

abundance of the phylum Bacteroidetes and Actino- upper jejunal digesta, the relative abundance of the fam-
bacteria was decreased due to the reduced dietary Ca ily Staphylococcus was decreased by the reduced dietary
and P level with the supplementation of 25-OHD3 Ca and P level with the supplementation of 25-OHD3
(interaction; P < 0.05). (interaction; P < 0.05).
Relative abundance of microbial composition at the Relative abundance of microbial composition at the
family level in the upper jejunal digesta of broiler chick- phylum level in the microbiota of the cecal digesta of
ens infected with Eimeria spp. and fed different levels of broiler chickens infected with Eimeria spp. and fed dif-
Ca, P, and 25-OHD3 is shown in Table 9. Eimeria chal- ferent levels of Ca, P, and 25-OHD3 is shown in Table 10
lenging significantly decreased the relative abundance of and Figure 4. In the microbiota of the cecal digesta, the
the family Peptostreptococcaceae, Burkholderiaceae, relative abundance of the phylum Proteobacteria was
Bacillaceae, Lachnospiraceae, Christensenellaceae, significantly increased by Eimeria challenging. In the
Staphylococcaceae, and Ruminococcaceae in the micro- microbiota of the cecal digesta, the relative abundance
biota of the upper jejunal digesta. There were interac- of the phylum Bacteroidetes tended to be increased by
tions between the dietary Ca and P level and Eimeria challenging (P = 0.068). The supplementation
supplementation of 25-OHD3 in the Eimeria challenging of 25-OHD3 significantly decreased the relative abun-
condition in the relative abundance of family Enterobac- dance of Bacteroidetes in the microbiota of the cecal
teriaceae in the microbiota of the upper jejunal digesta digesta. The Eimeria challenging tended to decrease the
(P < 0.05). The reduced level of dietary Ca and P signifi- relative abundance of the phylum Cyanobacteria in the
cantly decreased the relative abundance of the family microbiota of the cecal digesta (P = 0.054).
Burkholderiaceae in the upper jejunal microbiota. There Relative abundance of microbial composition at the
tended to be interactions between the level of dietary Ca family level in the upper jejunal digesta of broiler chick-
and P and Eimeria infection in the relative abundance of ens infected with Eimeria spp. and fed different levels of
the family Burkholderiaceae in the upper jejunal micro- Ca, P, and 25-OHD3 is presented in Table 11. Eimeria
biota (P = 0.084). The supplementation of 25-OHD3 challenging significantly increased the relative abun-
tended to increase the relative abundance of the family dance of the family Enterobacteriaceae in the micro-
Streptococcaceae in the non-challenging condition. The biota of the cecal digesta. Eimeria challenging
relative abundance of the family Ruminococcaceae significantly decreased the relative abundance of the
tended to be increased due to the reduction in the die- family Ruminococcaceae, Christensenellaceae, Lachno-
tary Ca and P level (P = 0.074). In the microbiota of the spiraceae, and Erysipelotrichaceae in the microbiota of
Table 7. Effects of the reduced dietary calcium (Ca) and phosphorous (P) level with the supplementation of 25-hydroxycholecalciferol (25-OHD3) on the alpha diversity of the jejunal and
cecal microbiota in broiler chickens in the general and Eimeria challenging condition.1

Jejunum Ceca

MICROBIOTA WITH DIETATY CA, P, AND 25-OHD3

Faith Faith
Pielou’s phylogenetic Observed Shannon Pielou’s phylogenetic Observed Shannon
E Ca&P 25-OHD3 evenness diversity features entropy evenness diversity features entropy
T1 NC N N 0.445 18.35 207.2ab 3.432 0.647a 17.867ab 258.0abc 5.184a
T2 NC L N 0.503 22.71 325.8a 4.221 0.612ab 18.927a 282.2a 4.991ab
T3 NC N S 0.561 26.25 256.5ab 4.361 0.676a 19.243a 281.3a 5.501a
T4 NC L S 0.450 22.32 275.7ab 3.639 0.692a 18.608a 277.7ab 5.609a
T5 C N N 0.389 20.83 216.8ab 3.036 0.377c 15.473b 207.0c 2.911c
T6 C L N 0.438 17.08 187.2b 3.279 0.411c 15.401b 220.5abc 3.211c
T7 C N S 0.407 21.66 244.2ab 3.232 0.452bc 15.362b 203.8c 3.468bc
T8 C L S 0.333 18.51 203.0ab 2.580 0.424bc 15.173b 214.7bc 3.289c
SEM2 0.138 5.138 69.14 1.169 0.106 1.654 34.56 0.893
P value
Treatments3 0.189 0.081 0.024 0.181 <.0001 <.0001 <.0001 <.0001
E 0.019 0.059 0.011 0.013 <.0001 <.0001 <.0001 <.0001
Ca&P 0.632 0.281 0.406 0.802 0.915 0.933 0.268 0.971
25-OHD3 0.884 0.108 0.599 0.909 0.117 0.709 0.807 0.136
E £ Ca&P 0.861 0.224 0.013 0.726 0.841 0.721 0.924 0.843
E £ 25-OHD3 0.350 0.381 0.585 0.532 0.863 0.469 0.490 0.773
Ca&P £ 25-OHD3 0.075 0.203 0.172 0.082 0.929 0.348 0.449 0.864
E £ Ca&P £ 25-OHD3 0.774 0.142 0.277 0.650 0.360 0.414 0.532 0.453
1
E: Eimeria challenging (oral gavage of water or oral gavage of 125,000 Eimeria acervulina, 25,000 Eimeria maxima, and 25,000 Eimeria tenella on D 14), NC: Eimeria non-challenged, C: Eimeria challenged,
Ca&P: dietary calcium and phosphorus levels (0.84% Ca and 0.42% available P or 0.64% Ca and 0.24% available P), N: normal dietary Ca and P level, R: reduced dietary Ca and P level, 25-OHD3: supplementa-
tion of 25-OHD3 (3,000 IU/kg), NS: 25-OHD3 non-supplemented, S: 25-OHD3 supplemented, T: treatment. All groups were compared using PROC MIXED based on a 2 £ 2 £ 2 factorial design with main effects
of Eimeria challenging (E), dietary Ca and P level (Ca&P), and 25-OHD3 supplementation (25-OHD3). The interactions (E £ Ca&P, E £ 25-OHD3, Ca&P £ 25-OHD3, and E £ Ca&P £ 25-OHD3) among the
main effects were investigated. Different letters within the same column indicates significant differences according to the Tukey’s HSD (honestly significant difference) test (P < 0.05).
2
SEM: Standard error of the mean.
3
P values for all treatments comparison.

9
10 CHOI ET AL.

Figure 3. Interactions between Eimeria challenging (E), the dietary calcium (Ca) and phosphorous (P) levels (Ca&P), and the supplementa-
tion of 25-hydroxycholecalciferol (25-OHD3) on the alpha diversity of the jejunal microbiota in broiler chickens. Selected parameters with statisti-
cal differences (P < 0.10) in interactions are shown. Different letters indicate statistically significant differences (P < 0.05). NC: Eimeria non-
challenged; C: Eimeria challenged; N: normal dietary Ca and P level; R: reduced dietary Ca and P level; NS: 25-OHD3 non-supplemented, S: 25-
OHD3 supplemented.

Table 8. Effects of the reduced dietary calcium (Ca) and phosphorous (P) level with the supplementation of 25-hydroxycholecalciferol
(25-OHD3) on the relative taxa abundance (%) at the phylum level in the jejunal microbiota in broiler chickens in the general and Eime-
ria challenging condition.1

E Ca&P 25-OHD3 Firmicutes Cyanobacteria Proteobacteria Bacteroidetes Actinobacteria Unassigned

ab
T1 NC N N 57.08 30.72 8.56 1.084 0.766 1.784
T2 NC L N 48.25 22.97 23.89 2.087 0.875ab 1.935
T3 NC N S 67.31 8.97 17.15 1.688 1.436a 3.449
T4 NC L S 56.75 29.47 10.52 1.255 0.433b 1.577
T5 C N N 36.19 10.92 20.71 1.174 0.388b 30.62
T6 C L N 54.86 7.82 4.80 2.092 0.447b 29.98
T7 C N S 31.55 8.62 5.49 1.163 0.692ab 52.49
T8 C L S 36.88 9.69 34.16 0.846 0.372b 18.05
SEM2 27.41 17.71 22.56 0.912 0.469
P value
Treatments3 0.291 0.101 0.305 0.160 0.005
E 0.033 0.010 0.848 0.431 0.005
Ca&P 0.885 0.603 0.415 0.272 0.039
25-OHD3 0.903 0.448 0.721 0.166 0.402
E £ Ca&P 0.178 0.474 0.877 0.976 0.248
E £ 25-OHD3 0.199 0.473 0.472 0.334 0.998
Ca&P £ 25-OHD3 0.637 0.121 0.391 0.015 0.009
E £ Ca&P £ 25-OHD3 0.716 0.246 0.015 0.851 0.183
1
E: Eimeria challenging (oral gavage of water or oral gavage of 125,000 Eimeria acervulina, 25,000 Eimeria maxima, and 25,000 Eimeria tenella on D
14), NC: Eimeria non-challenged, C: Eimeria challenged, Ca&P: dietary calcium and phosphorus levels (0.84% Ca and 0.42% available P or 0.64% Ca
and 0.24% available P), N: normal dietary Ca and P level, R: reduced dietary Ca and P level, 25-OHD3: supplementation of 25-OHD3 (3,000 IU/kg), NS:
25-OHD3 non-supplemented, S: 25-OHD3 supplemented, T: treatment. All groups were compared using PROC MIXED based on a 2 £ 2 £ 2 factorial
design with main effects of Eimeria challenging (E), dietary Ca and P level (Ca&P), and 25-OHD3 supplementation (25-OHD3). The interactions
(E £ Ca&P, E £ 25-OHD3, Ca&P £ 25-OHD3, and E £ Ca&P £ 25-OHD3) among the main effects were investigated. Different letters within the same
column indicates significant differences according to the Tukey’s HSD (honestly significant difference) test (P < 0.05).
2
SEM: Standard error of the mean
3
P values for all treatments comparison
 MICROBIOTA WITH DIETATY CA, P, AND 25-OHD3 11

Figure 4. Interactions between Eimeria challenging (E), the dietary calcium (Ca) and phosphorous (P) levels (Ca&P), and the supplementa-
tion of 25-hydroxycholecalciferol (25-OHD3) on the taxa abundance of the jejunal and cecal microbiota in broiler chickens. Selected parameters
with statistical differences (P < 0.10) in interactions are shown. Different letters indicate statistically significant differences (P < 0.05). NC: Eimeria
non-challenged; C: Eimeria challenged; N: normal dietary Ca and P level; R: reduced dietary Ca and P level; NS: 25-OHD3 non-supplemented, S:
25-OHD3 supplemented.

the cecal digesta. The supplementation of 25-OHD3 sig- consistent with previous studies indicating that Eimeria
nificantly increased the relative abundance of the family challenging modulated the expression of antimicrobial
Ruminococcaceae in the microbiota of the cecal digesta. peptides in the gastrointestinal tract (Choi and Kim,
The supplementation of 25-OHD3 increased the relative 2022; Goo et al., 2023). However, there were variations
abundance of the family Ruminococcaceae more dra- in the trends, with some showing an increase and others
matically in the non-challenging condition compared to reporting a decrease in the expression of antimicrobial
the Eimeria challenging condition (interaction; P < peptides under the Eimeria challenging condition. These
0.05) findings suggest that Eimeria challenging can lead to a
decrease in the expression of antimicrobial peptides,
while simultaneously inducing overexpression of antimi-
DISCUSSION crobial peptides as a defensive mechanism in birds (Su et
al., 2017). Interestingly, the mRNA expression of VDR
The purpose of the study was to investigate the effects in the ceca was upregulated by Eimeria challenging in
of the reduced dietary Ca and P level and supplementa- the current study. Several studies showed that the
tion of 25-OHD3 on the expression of VDR, antimicro- decreased expression of VDR is closely associated with
bial peptides, and TLRs, activities of brush border intestinal diseases and inflammation in the large intes-
digestive enzymes, antioxidant capacity, and microbiota tine in human models (Kong et al., 2008; Kim et al.,
in the upper jejunum and ceca of broiler chickens with/ 2013; Li et al., 2015). These results potentially suggest
without Eimeria challenge. In the current study, Eime- that VDR may play an important role in regulating
ria challenging increased the expression of antimicrobial immune systems in the ceca of broiler chickens. In the
peptides including AvBD3, AvBD9, and CATH3 in the current study, Eimeria challenging negatively affected
jejunum and ceca, whereas the expression of LEAP2 was the cecal microbiota, which is consistent with our previ-
reduced in the jejunum and ceca. These results are ous studies (Choi et al., 2023a). Potentially, the
12 CHOI ET AL.

of Eimeria challenging (E), dietary Ca and P level (Ca&P), and 25-OHD3 supplementation (25-OHD3). The interactions (E £ Ca&P, E £ 25-OHD3, Ca&P £ 25-OHD3, and E £ Ca&P £ 25-OHD3) among the
Table 9. Effects of the reduced dietary calcium (Ca) and phosphorous (P) level with the supplementation of 25-hydroxycholecalciferol (25-OHD3) on the relative taxa abundance (%) at

Unassigned

tion of 25-OHD3 (3,000 IU/kg), NS: 25-OHD3 non-supplemented, S: 25-OHD3 supplemented, T: treatment. All groups were compared using PROC MIXED based on a 2 £ 2 £ 2 factorial design with main effects
E: Eimeria challenging (oral gavage of water or oral gavage of 125,000 Eimeria acervulina, 25,000 Eimeria maxima, and 25,000 Eimeria tenella on D 14), NC: Eimeria non-challenged, C: Eimeria challenged,
Ca&P: dietary calcium and phosphorus levels (0.84% Ca and 0.42% available P or 0.64% Ca and 0.24% available P), N: normal dietary Ca and P level, R: reduced dietary Ca and P level, 25-OHD3: supplementa-
increased relative abundance of the phylum Proteobac-

68.91
58.15
42.89
58.74
74.63
89.17
89.56
62.67
teria and the decreased activities of serum alkaline phos-
phatase activities would result in an increase on serum
endotoxin level in broiler infected with Eimeria spp. in
Ruminococcaceae

the present study. This is because alkaline phosphatase,

0.868
2.080
1.381
1.534
0.697
1.275
0.792
0.841
0.941

0.185
0.044
0.074
0.733
0.501
0.780
0.152
0.629
which is activated by short chain fatty acids, has an
important role in detoxifying endotoxins (Koyama and
Ono, 1976; Chen et al., 2010).
Sphingomonadaceae

In the current study, the upper jejunum was selected

main effects were investigated. Different letters within the same column indicates significant differences according to the Tukey’s HSD (honestly significant difference) test (P < 0.05).
for analysis because most of vitamins, Ca, and phos-
0.282
0.296
0.847
0.119
0.477
0.148
0.388
0.328
0.604

0.547
0.772
0.122
0.496
0.645
0.672
0.502
0.155
phate are absorbed in the upper gastrointestinal tract in
birds (Hurwitz and Bar, 1971; Favus, 1985). It was
hypothesized that the major alterations on gut ecosys-
Staphylococcaceae

tems by 25-OHD3, Ca, and P should be observed in the

upper gastrointestinal tract rather than the lower gas-
0.581
0.542
0.718
0.244
0.062
0.361
0.196
0.025
0.435

0.075
0.007
0.449
0.474
0.209
0.938
0.079
0.945

trointestinal tract in broiler chickens, which is supported

by a previous by Bashir et al. (2016). Although the
upper jejunum is not the main area for microbial activi-
Christensenellaceae

ties, the upper jejunal microbiota would be closely asso-

3.727ab

4.424ab
4.120ab

1.922ab

1.828ab

ciated to gut ecosystem and gut inflammation in the

1.506b

1.395b
6.609a

2.725

0.020
<0.001
0.282
0.529
0.586
0.617
0.320
0.315

upper gastrointestinal tract, which is the area for nutri-

ent digestion and absorption in broiler chickens (El Aidy
et al., 2015; Zhang et al., 2022). Furthermore, alteration
Lachnospiraceae

of the microbiota in the upper small intestine can influ-

4.049
3.576
5.579
2.055
1.058
1.250
1.289
0.750
3.205

0.117
0.005
0.247
0.944
0.330
0.941
0.313
0.534

ence the microbiota in the large intestine. The ceca, the

the family level in the jejunal microbiota in broiler chickens in the general and Eimeria challenging condition.1

main area for microbial fermentation with the higher

density of microbes, was also analyzed in the current
study.
Bacillaceae

1.779
1.949
1.770
2.086
1.216
0.657
0.944
0.359
1.397

0.287
0.009
0.686
0.786
0.319
0.667
0.941
0.915

In the current study, the reduced dietary Ca and P

level decreased the relative abundance of TLRs includ-
ing TLR3 and TLR5 and antimicrobial peptides includ-
Streptococcaceae

ing AvBD5, AvBD9, and CATH3 in the Eimeria

1.193
0.959
2.368
4.748
1.048
1.348
1.508
0.549
2.752

0.208
0.131
0.636
0.146
0.374
0.097
0.666
0.222

challenging condition. Downregulated antimicrobial

peptides and TLRs in the Eimeria challenging condition
may indicate that defensive mechanisms against Eime-
ria challenging were not properly activated. In consis-
Burkholderiaceae

tent, a previous study by Hofmann et al. (2021) showed

3.422ab
1.170ab

0.890ab
0.779ab

1.107ab
0.587b

0.387b
10.843a

5.518

0.033
0.041
0.046
0.261
0.084
0.278
0.204
0.267

that the reduction of dietary Ca and P could compro-

mise the immune system of birds. In the present study,
although statistical differences were not obtained, the
Peptostreptococcaceae

reduced levels of dietary Ca and P numerically downre-

gulated VDR in the upper jejunum, which is consistent
12.448

26.051
18.900
8.101

0.185
0.087
0.177
0.222

0.255
0.012
0.640
0.324
0.644
0.329
0.914
0.905
21.25

with a previous study by Rodriguez-Lecompte et al.

(2016). VDR is known to regulate diverse functions of
immune system of animals (Shojadoost et al., 2015; Bat-
tistini et al., 2020) and the expression of antimicrobial
peptides (Gombart, 2009), which can modulate the
Enterobacteriaceae

microbiota of broiler chickens (Zong et al., 2020). Poten-

0.226
0.293
0.224
0.890
0.907
0.378
0.189
0.038

P values for all treatments comparison.

2.74

3.14
6.57

3.20
2.65
16.56

18.34

32.04
22.11

tially, the lack of defensive mechanisms in the gut due to

SEM: Standard error of the mean.

the reduced dietary Ca and P level in the Eimeria chal-

lenging condition resulted in a decrease observed fea-
tures in the current study, which is considered as
25-OHD3

unfavorable for the gut microbiota (Aruwa et al., 2021).

N
N

N
N
S
S

S
S

In the present study, the activities of upper jejunal malt-

ase were decreased due to the reduced dietary Ca and P
Ca&P

level in the Eimeria challenging condition. Eimeria chal-

E £ Ca&P £ 25-OHD3
N

N
L

Ca&P £ 25-OHD3

lenging condition negatively altered the absorption and

E £ 25-OHD3

metabolism of dietary Ca and P (Tompkins et al., 2023),

Treatments3
NC
NC
NC
NC
C
C
C
C
E

E £ Ca&P
25-OHD3

which would explain that compromised gut ecosystem

P value

Ca&P
SEM2

2
3

by the reduced dietary Ca and P level were mainly

T1
T2
T3
T4
T5
T6
T7
T8

E
 MICROBIOTA WITH DIETATY CA, P, AND 25-OHD3 13
Table 10. Effects of the reduced dietary calcium (Ca) and phosphorous (P) level with the supplementation of 25-hydroxycholecalciferol
(25-OHD3) on the relative taxa abundance (%) at the phylum level in the cecal microbiota in broiler chickens in the general and Eime-
ria challenging condition1

E Ca&P 25-OHD3 Proteobacteria Bacteroidetes Firmicutes Cyanobacteria Actinobacteria Unassigned

T1 NC N N 12.20ab 18.92 65.30a 0.092 0.813 2.673
T2 NC L N 18.15ab 18.02 59.98a 0.064 1.000 2.793
T3 NC N S 8.56b 16.05 69.00a 0.127 0.996 5.268
T4 NC L S 6.67b 12.78 75.79a 0.104 0.924 3.735
T5 C N N 44.20ab 29.31 24.21b 0.054 0.456 1.778
T6 C L N 32.08ab 34.60 31.02b 0.087 0.579 1.622
T7 C N S 45.50ab 18.23 32.18b 0.062 0.759 3.272
T8 C L S 50.66a 16.82 29.42b 0.049 0.913 2.137
SEM2 21.22 15.31 14.15 0.448 0.934 1.667
P value
Treatments3 <0.001 0.232 <0.001 0.050 0.338
E <.0001 0.068 <.0001 0.054 0.290
Ca&P 0.907 0.987 0.737 0.453 0.356
25-OHD3 0.847 0.043 0.121 0.157 0.340
E £ Ca&P 0.656 0.651 0.875 0.757 0.367
E £ 25-OHD3 0.161 0.247 0.426 0.312 0.329
Ca&P £ 25-OHD3 0.702 0.611 0.878 0.662 0.313
E £ Ca&P £ 25-OHD3 0.312 0.808 0.192 0.581 0.304
1
E: Eimeria challenging (oral gavage of water or oral gavage of 125,000 Eimeria acervulina, 25,000 Eimeria maxima, and 25,000 Eimeria tenella on D
14), NC: Eimeria non-challenged, C: Eimeria challenged, Ca&P: dietary calcium and phosphorus levels (0.84% Ca and 0.42% available P or 0.64% Ca
and 0.24% available P), N: normal dietary Ca and P level, R: reduced dietary Ca and P level, 25-OHD3: supplementation of 25-OHD3 (3,000 IU/kg), NS:
25-OHD3 non-supplemented, S: 25-OHD3 supplemented, T: treatment. All groups were compared using PROC MIXED based on a 2 £ 2 £ 2 factorial
design with main effects of Eimeria challenging (E), dietary Ca and P level (Ca&P), and 25-OHD3 supplementation (25-OHD3). The interactions
(E £ Ca&P, E £ 25-OHD3, Ca&P £ 25-OHD3, and E £ Ca&P £ 25-OHD3) among the main effects were investigated. Different letters within the same
column indicates significant differences according to the Tukey’s HSD (honestly significant difference) test (P < 0.05).
2
SEM: Standard error of the mean
3
P values for all treatments comparison

observed in the Eimeria challenging condition in the results, the supplementation of 5,000 IU/kg 25-OHD3
current study. Nevertheless, the reduced level of dietary may not be enough to regulate the gene expression of
Ca and P decreased the relative abundance of the family antimicrobial peptides and TLRs. Nonetheless, the
Burkholderiaceae, a potential pathogenic family group supplementation of 25-OHD3 beneficially modulated
(Proszkowiec-Weglarz et al., 2020), in the jejunal micro- the cecal microbiota by decreasing the relative abun-
biota in the non-challenging condition and increased the dance of the phylum Bacteroidetes and increasing the
relative abundance of the family Ruminococcaceae, an relative abundance of the family Ruminococcaceae. A
important group for producing SCFA (Acharya et al., previous study by Choi et al. (2022a) showed that
2022). These results showed that the reduced level of higher relative abundance of the phylum Bacteroidetes
dietary Ca and P could inhibit or augment the growth of are associated with lower growth performance in broiler
specific microbes. Nonetheless, these differences by the chickens. The higher relative abundance of the family
reduced dietary Ca and P level did not result in the mod- Ruminococcaceae, an important group for producing
ulation of the cecal microbiota in the current study. SCFA (e.g., important energy sources for hosts), can
Overall, the reduced dietary Ca and P level modulated be beneficial to improve growth performance broiler
mucosal immunity and microbiota in the upper jejunum chickens (Diaz Carrasco et al., 2018; Choi et al., 2021).
of broiler chickens in the non-challenging and Eimeria Along with this, the supplementation of 25-OHD3
challenging condition. improved activities of lipase in the upper jejunum, and
Whereas the level of dietary Ca and P is thought to the supplementation of 25-OHD3 decreased the serum
modulate gut ecosystem mainly via altering the expres- endotoxin level in the Eimeria challenging condition in
sion of VDR in the current study, the supplementation the current study. These results would be associated
of 25-OHD3 did not alter the expression of VDR and beneficially modulated gut mucosa layer and gut integ-
antimicrobial peptides in the upper jejunum and ceca rity due to the supplementation of 25-OHD3. Akimbe-
in the current study. A previous study by Capiati et al. kov et al. (2020) demonstrated that vitamin D has an
(2002) demonstrated that 25-OHD3 can regulate gene important role in thickening gut mucosa layer. Poten-
expression at the cellular level. However, no studies tially, the supplementation of 25-OHD3 thickened the
have shown that feed supplementation of 25-OHD3 can mucosa layer, which increased the relative abundance
regulate the expression of VDR in the gastrointestinal of the beneficial bacterial group (e.g., family Rumino-
tract of broiler chickens. A previous study by Rodri- coccaceae), trapped more lipase (pancreatic enzyme),
guez-Lecompte et al. (2016) showed the supplementa- and improved gut barrier integrity to decrease the per-
tion of 25-OHD3 ranging from 200 to 9,800 IU/kg meation of endotoxin into blood circulation. Therefore,
modulated expression of TLRs, cytokines, and antimi- the supplementation of 25-OHD3 improved the gut
crobial peptides in broiler chickens. Based on the microbiota and gut barrier integrity in broiler chickens
14 CHOI ET AL.
Table 11. Effects of the reduced dietary calcium (Ca) and phosphorous (P) level with the supplementation of 25-hydroxycholecalciferol (25-OHD3) on the relative taxa abundance (%) at

Unassigned

of Eimeria challenging (E), dietary Ca and P level (Ca&P), and 25-OHD3 supplementation (25-OHD3). The interactions (E £ Ca&P, E £ 25-OHD3, Ca&P £ 25-OHD3, and E £ Ca&P £ 25-OHD3) among the
tion of 25-OHD3 (3,000 IU/kg), NS: 25-OHD3 non-supplemented, S: 25-OHD3 supplemented, T: treatment. All groups were compared using PROC MIXED based on a 2 £ 2 £ 2 factorial design with main effects
E: Eimeria challenging (oral gavage of water or oral gavage of 125,000 Eimeria acervulina, 25,000 Eimeria maxima, and 25,000 Eimeria tenella on D 14), NC: Eimeria non-challenged, C: Eimeria challenged,
Ca&P: dietary calcium and phosphorus levels (0.84% Ca and 0.42% available P or 0.64% Ca and 0.24% available P), N: normal dietary Ca and P level, R: reduced dietary Ca and P level, 25-OHD3: supplementa-
in general or under Eimeria challenging condition with-

43.44
40.58
42.53
38.39
37.61
46.76
32.29
31.42
out influencing the mRNA expression of VDR and anti-
Planococcaceae microbial peptides. Further studies are needed to
elucidate the mode of actions.
The interactions between dietary Ca and P level and
0.039
0.111
0.127
0.073
0.357
0.078
0.013
0.038
0.321

0.677
0.718
0.529
0.373
0.466
0.248
0.632
0.251
25-OHD3 are well documented in terms of absorption
and their effects. However, their interactive effects on
Sphingomonadaceae

the gut microbiota were not well documented yet. In the

current study, the reduced level of dietary Ca and P

main effects were investigated. Different letters within the same column indicates significant differences according to the Tukey’s HSD (honestly significant difference) test (P < 0.05).
with the supplementation of 25-OHD3 decreased shan-
0.118
0.210
0.246
0.239
0.054
0.267
0.217
0.287
0.299

0.884
0.969
0.294
0.332
0.568
0.941
0.489
0.899
non entropy (richness and evenness) of the upper jejunal
microbiota. Moreover, in the present study, unfavorable
Erysipelotrichaceae

jejunal bacterial groups including the phylum Bacteroi-

detes and family Staphylococcus was increased by the
reduced dietary Ca and P level without the 25-OHD3
0.204
0.487
0.400
0.312
0.171
0.170
0.150
0.252
0.200

0.051
0.007
0.207
0.721
0.684
0.865
0.254
0.046

supplementation but was decreased by the reduced die-

tary Ca and P level with the 25-OHD3 supplementation.
Peptostreptococcaceae

While the activities of sucrase in the upper jejunum, one

of the parameters for gut development, were decreased
due to the normal level of dietary Ca and P with 25-
0.378
0.309
0.458
0.197
0.336
0.236
0.292
0.658
0.394

0.575
0.696
0.888
0.452
0.198
0.373
0.549
0.155

OHD3 supplementation, they were increased due to the

reduced level of dietary Ca and P level with 25-OHD3
Streptococcaceae

supplementation. These results suggest that the effects

of the reduced dietary Ca and P level on the microbiota
0.680
0.693
1.036
1.456
1.573
1.211
1.345
0.801
1.005

0.649
0.364
0.685
0.681
0.256
0.138
0.848
0.614

and gut development in the upper intestine can be mod-

ulated by the supplementation of 25-OHD3 while it is
the family level in the cecal microbiota in broiler chickens in the general and Eimeria challenging condition.1
Bacillaceae

hard to determine whether it is beneficial or detrimental

1.339
1.134
1.555
0.681
0.867
1.024
1.270
0.730
1.261

0.922
0.577
0.321
0.931
0.636
0.814
0.354
0.985

effects on broiler chickens.

In conclusion, the reduced dietary Ca and P level
Lachnospiraceae

affected jejunal microbiota potentially via modulating

mucosal immunity in the upper jejunum of broiler chick-
<0.001
6.337
8.726
5.489
7.524
1.717
1.397
2.883
3.099
4.017

0.017

0.357
0.861
0.335
0.295
0.969
0.849

ens in the Eimeria challenging condition. The supple-

mentation of 25-OHD3 improved the gut microbiota and
Christensenellaceae

gut barrier integrity in broiler chickens with/without

Eimeria challenge. The dietary Ca and P level and 25-
OHD3 level can exhibit interactive effects on the gut
20.49abc

19.86abc
21.71ab

10.04bc

<0.001
<0.001
25.96a

9.93bc

9.98bc
6.711

0.440
0.514
0.274
0.786
0.424
0.656
8.59c

development and gut microbiota in broiler chickens.

Therefore, optimizing dietary Ca, P, and 25-OHD3 lev-
Ruminococcaceae

els is crucial for maintaining gut microbiota and health

in broiler chickens, especially when combating Eimeria
14.70ab

15.60ab

<0.001
<0.001
10.55b

26.16a

6.652

0.650
0.035
0.233
0.041
0.382
0.005
5.25b
7.76b
9.34b
3.93b

infection.
Enterobacteriaceae

DISCLOSURES
11.04ab
16.71ab

43.47ab
31.17ab
42.16ab

<0.001
48.80a
20.95

0.001

0.951
0.990
0.686
0.188
0.628
0.286
6.61b
5.11b

The authors declare no conflicts of interest.

25-OHD3

P values for all treatments comparison

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N
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