Effects of 25-hydroxycholecalciferol on performance, gut health, and bone
quality of broilers fed with reduced calcium and phosphorus diet during Eimeria
challenge
T. S. B. Lopes ,* H. Shi,y D. White,y I. C. S. Ara

ujo ,* and W. K. Kim
*
Department of Animal Science, Universidade Federal de Minas Gerais, Belo Horizonte, MG 31270-901, Brazil; and
y
Department of Poultry Science, University of Georgia, Athens, GA 30602, USA
ABSTRACT This study evaluated the effects of 25-
hydroxycholecalciferol (25-OHD) on performance, gut
health, and bone quality of broilers fed with reduced cal-
cium (Ca) and phosphorus (P) diet during Eimeria spp.
challenge. A total of 576 fourteen-day-old Cobb 500 male
chicks were randomly distributed in a 2 £ 2 £ 2 factorial
arrangement, with 6 replicates of 12 birds each. The main
factors were 25-OHD level (0 or 3,000 IU/kg of feed),
mineral level (0.84% of Ca/0.42% of P, the levels recom-
mended for the grower phase (NOR) or 0.64% of Ca/
0.22% of P (RED), and mid-high mixed Eimeria chal-
lenge or nonchallenge. 25-OHD improved phosphorus
Key words: broiler, 25-hydroxycholecalciferol, Eimeria, bone microstructure, mineral reduction
INTRODUCTION
Eimeria is a genus of protozoans that can parasitize
different livestock species, including poultry (Blake et
al., 2021). Regarding broilers, coccidiosis has been listed
as one of the most important diseases, causing a global
economic loss of about 14 to 12 billion dollars per year to
the broiler industry (Blake et al., 2020). Eimeria acervu-
lina, Eimeria maxima, and Eimeria tenella can invade
the duodenum, jejunum and ceca, respectively, causing
intestinal damage, which severely impairs nutrient
absorption and performance (Teng et al., 2020, 2023;
Choi et al., 2021). Recent studies showed that Eimeria
spp. infection can reduce the absorption of calcium (Ca)
and phosphorus (P), leading to poor bone mineraliza-
tion (Sakkas et al., 2018; Shi et al., 2022) and an increase
in bone reabsorption due to an enhance in circulating
RANKL, a protein responsible for activating the
Ó 2023 The Authors. Published by Elsevier Inc. on behalf of Poultry
Science Association Inc. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/
4.0/).
Received July 15, 2023.
Accepted November 3, 2023.
1
Corresponding author: wkkim@uga.edu
1
y,1
retention (P = 0.019), bone ash weight (P = 0.04), corti-
cal bone trabecular connectivity (P = 0.043) during coc-
cidiosis. For birds fed with reduced mineral levels, 25-
OHD supplementation increased bone ash weight
(P = 0.04). However, 25-OHD did not improve bone ash
weight when birds were challenged and fed with reduced
mineral levels. The dietary 3,000 IU of 25-OHD supple-
mentation did not improve performance or gut morphol-
ogy but support bone health during coccidiosis. Future
investigations are needed for better understand 25-OHD
role on bone microarchitecture and oxidative metabolism
during coccidiosis.
2024 Poultry Science 103:103267
https://doi.org/10.1016/j.psj.2023.103267
osteoclast differentiation cascade (Kakhki et al., 2019;
Tompkins et al., 2023).
Vitamin D3 (VitD), a fat-soluble vitamin synthesized
endogenously from 7-dehydrocholesterol, can also be
supplemented in the feed, in the forms of cholecalciferol,
25-hydroxycholecalciferol, or 1,25-Dihydroxyvitamin D3
(Soares Jr. et al., 1995). The VitD is responsible for the
active Ca (Wasserman et al., 1992) and P (Shao et al.,
2019) absorption pathways in the intestine, renal Ca
reabsorption (Jones et al., 1998), and development and
remodeling of bone tissue (DeLuca, 1988). The VitD use
in broiler diets has been linked to improved feed intake,
weight gain, feed conversion (Han et al., 2017), bone per-
centage of ash, and breaking strength (Leyva-Jimenez et
al., 2019). VitD can reduce the occurrence of bone
pathologies, such as tibial dyschondroplasia (Khan et
al., 2010), especially in situations of unbalance in cal-
cium and phosphorus diet levels (Zhang et al., 2020). In
the present study the VitD as feed additive in its 25-
hydroxycholecalciferol form will be refer as 25-OHD.
There is a gap in the knowledge regarding the use of 25-
OHD in the diet of broilers infected with Eimeria spp.,
especially to the effects on bone quality and gut health
during a severe restriction of dietary minerals. Thus, the
aim of this study was to evaluate the effects of VitD sup-
plementation in the form of 25-OHD to a Ca and P
2 LOPES ET AL.
reduced diet on performance, bone quality, and gut
health of broilers infected with E. acervulina, E. maxima,
and E. tenella.
MATERIALS AND METHODS
General Procedures
An experiment was conducted under the approval of
the Institutional Animal Care and Use Committee of
University of Georgia (Athens, GA). A total of 576 four-
teen-day-old male Cobb 500 broiler chicks were distrib-
uted in a completely randomized design with a factorial
arrangement 2 £ 2 £ 2, with 6 replicates of 12 birds
each. The main effects were the inclusion of 25-OHD (0
IU or 3,000 IU/kg of feed), calcium and phosphorus lev-
els (0.84% of Ca and 0.42% of available P, NOR or
0.64% of Ca and 0.22% of available P, RED), and path-
ogen inoculation (challenged with or without a pool of
Eimeria acervulina, Eimeria maxima, and Eimeria ten-
ella). The chicks were allocated in cages equipped with a
feed and a nipple drinker, providing ad libitum access to
water and feed from 1 to 26 d of age. Temperature and
lighting program followed the recommendation of Cobb
500 management guide (Cobb Vantress, 2018a).
The diets were based on corn and soybean meal and
formulated to meet or exceed the nutritional levels for
the starter and grower phases (Cobb Vantress, 2018b),
except for 25-OHD, Ca, and P in the grower diets
(Table 1). The grower diets included 0.3% of Titanium
dioxide (TiO2, Sigma Aldrich, St. Louis, MO) as an indi-
gestible marker for determination of total tract retention
of nutrients. The treatments were as follows: 1) 0 IU of
25-OHD/kg of feed and 0.84% of Ca and 0.42% of avail-
able P, no challenge; 2) 0 IU of 25-OHD/kg of feed and
0.64% of Ca and 0.22% of available P, no challenge; 3)
3,000 IU of 25-OHD/kg of feed, NOR, no challenge; 4)
3,000 IU of 25-OHD/kg of feed, no challenge; 5) 0 IU of
25-OHD/kg of feed, NOR, with challenge; 6) 0 IU of 25-
OHD/kg of feed, RED, with challenge; 7) 3,000 IU of 25-
OHD/kg of feed, NOR, with challenge; and 8) 3,000 IU
of 25-OHD/kg of feed, RED, with challenge. All diets
included cholecalciferol that was present in the vitamin
premix.
On d 14, the birds in challenge groups (288 birds) were
orally gavaged with 1 mL of a solution containing
approximately 25,000 sporulated E. maxima oocysts,
25,000 sporulated E. tenella oocysts, and 125,000 sporu-
lated E. acervulina oocysts, whereas the other half was
gavaged with distilled water. The Eimeria spp. used in
the study were field isolates. The challenge dose was
determined by a previous study conducted at our lab
and was intended to cause mid-high coccidiosis infection
(Teng et al., 2020a).
The birds were weighed, by cage, at 14, 20, and 26 d of
the experiment. The feed was weighed daily, by cage,
starting at 14 d (0 dpi: 0 days postinoculation) until 26
d (12 dpi). Mortality was also recorded daily. The body
weight (BW), body weight gain (BWG), and feed
intake (FI) were determined from 14 to 20 d (0−6 dpi,
acute challenge phase) and from 21 to 26 d (7−12 dpi,
recovery phase). Feed conversion rate (FCR) was calcu-
lated and corrected for mortality.
Sample Collection and Analysis Performed
Total Tract Apparent Retention of Macrominerals TagedAPR-
APOven-dried feed and excreta from 6 dpi were ground to
measure Ca and P levels of feed and excreta were mea-
sured by the Soil Laboratory, University of Georgia.
The titanium dioxide was analyzed according to Short
et al. (1996). Briefly, 0.25 g of excreta samples or 1.0 g of
feed samples were ashed in a porcelain crucible at 600°C
overnight to burn out organic materials. After cooling,
an additional 10 mL of a 7.4 M H2SO4 solution was
added to each crucible. The mixture was boiled for
approximately 60 min until completely dissolved. After
cooling, 25 mL of distilled water was added to each sam-
ple. The contents were poured in 100 mL volumetric
flasks through filter paper (Whatman 541), and 20 mL
of H2O2 (30%) was add to each flash. Then, the content
was diluted to 100 mL with distilled water and mixed
thoroughly by inverting the flasks. The concentration of
titanium dioxide was determined at 400 nm using a spec-
trophotometer (Spectramac M5, Molecular Devices, San
Jose, CA). The apparent total tract retention of miner-
als was calculated according to the following
equation (Adhikari et al., 2020):
   
Ti N0
ATTD; % ¼ 1    100
Ti0 Ni
where Ti represents the level of titanium in the diet; Ti0
represents the level of titanium in the excreta; Ni repre-
sents the determined value of the nutrient in the diet; N0
represents the concentration of the nutrient in the
excreta.
Intestinal Histomorphometry At 20 d (6 dpi), 1 bird/-
pen was euthanized by cervical dislocation, and portions
of approximately 2 cm of the duodenal loop, middle jeju-
num, and ileum were collected, flushed with 1£ PBS
(National Diagnostics, Atlanta, GA), and stored in 10%
neutral-buffered formalin until processed (Chen et al.,
2019). For the preparation of the histology slides, the tis-
sue samples were dehydrated in increasing concentra-
tions of ethanol, diaphanized in xylol, and embedded in
paraffin. Serial cuts of 4mm tissue slides were stained
with hematoxylin-eosin and analyzed in a light micro-
scope (BZ-X800E, Keyence Inc., Itasca, IL). Pictures
were taken and analyzed using an image analyzer soft-
ware (BZ-X 800 Analyzer, Keyence Inc., Itasca, IL) to
measure crypts depth and villi height of 9 crypts and 9
villi per slide. The ratio of villi height to crypts depth
was calculated from each sample.
Oocyst Shedding At 19 d (5 dpi), clean paper was
kept under each cage, and the excreta were collected for
24 h. From each cage, approximately 300 g of represen-
tative excreta was collected in sample bag and stored at
4°C until further processing following the modified pro-
cedure by Teng et al. (2020b). For processing, 5 g of
 25-HYDROXYCHOLECALCIFEROL IN BROILERS UNDER COCCIDIOSIS 3
Table 1. Feed formulation for broiler starter and grower diets (as-fed basis).

Grower (14−26)
Item1 Starter (0−14) NOR NOR + 25-OHD RED RED + 25-OHD
Corn, grain 58.52 58.00 58.00 60.00 60.00
Soybean meal 45% CP 35.65 33.02 33.02 32.69 32.69
Soybean oil 2.00 3.70 3.70 2.98 2.98
Dical. phos. 1.59 1.45 1.45 0.40 0.40
Limestone 1.14 1.08 1.08 1.18 1.18
Sand/product space 0.00 1.71 1.60 1.70 1.59
Common salt 0.40 0.34 0.34 0.34 0.34
DL-Methionine 0.30 0.22 0.22 0.22 0.22
L-Lysine HCL 0.16 0.00 0.00 0.01 0.01
L-Threonine 0.06 0.00 0.00 0.00 0.00
Vitamin premix2 0.10 0.10 0.10 0.10 0.10
Mineral premix3 0.08 0.08 0.08 0.08 0.08
Titanium dioxide 0.00 0.30 0.30 0.30 0.30
25-OHD 0.00 0.00 0.11 0.00 0.11
Calculated composition
ME (kcal/kg) 3.050 3.100 3.100 3.100 3.100
Crude protein (%) 21.52 20.00 20.00 20.00 20.00
Crude fiber (%) 2.18 2.09 2.09 2.12 2.12
Calcium (%) 0.90 0.84 (0.94)4 0.84 (0.75) 0.64 (0.61) 0.64 (0.82)
Available phosphorus (%) 0.45 0.41 (0.42)5 0.41 (0.41) 0.22 (0.25) 0.22 (0.25)
Digestible-Lys (%) 1.22 1.02 1.02 1.02 1.02
Digestible-Met (%) 0.62 0.52 0.52 0.52 0.52
Digestible-Treo (%) 0.83 0.72 0.72 0.72 0.72
Digestible-Trip (%) 0.26 0.24 0.24 0.24 0.24
25-OHD (IU/kg) 0.00 0.00 3,000 (3,080)6 0.00 3,000 (2,905)
1
Item: NOR: 0.84% of Ca. 0.42% of P; NOR + 25-OHD: 0.84% of Ca. 0.42% of P. and 3,000 IU of 25-hydroxycholecalcidiol/kg of feed; RED: 0.64% of
Ca. 0.22% of P; RED + 25-OHD: 0.64% of Ca. 0.22% of P. and 3,000 IU of 25-hydroxycholecalcidiol/kg of feed; 25-OHD: 25-hydroxycholecalciferol.
2
Provided per kg of vitamin premix: Vit. A 2,204,586 IU; cholecalciferol 440,917 IU; Vit E: 4,409 IU; Vit. B12 5 mg; biotin 44 mg. Menadione 441 mg.
Thiamine 882 mg; riboflavin 1,764 mg; d-pantothenic acid 4,409 mg; Vit. B6 882 mg; niacin 17,637 mg; folic acid 220 mg; choline 76,543 mg.
3
Provided per kg of mineral premix: Ca 0.72 g. Mn 3.04 g. Zn 2.43 g. Mg 0.61 g. Fe 0.59 g. Cu 22.68 g. I 22.68 g. Se 9.07 g.
4
Analyzed calcium levels.
5
Analyzed available phosphorus levels. Available phosphorus content was analyzed following the method by Latta and Eskin (1980).
6
Analyzed 25-dihydrxycholecalciferol levels.

excreta was diluted with 45 mL of water. After proper
mixing, 1 mL mixture was taken and diluted with 9 mL
of saturated salt solution. Homogenized mixtures were
incubated for 30 s to let oocysts float. Using a water
dropper pipette (Thermo Fisher Scientific, Waltham,
MA), the final samples were loaded in McMaster cham-
bers (Jorgensen Laboratories, Loveland, CO), and the
total oocysts were counted, and the data were calculated
as oocysts per gram using formula as shown by Yadav et
al. (2020).
Bone Ash On d 20 (6 dpi) and d 23 (9 dpi), 1 bird from
each pen was euthanized to collect tibia for measuring
bone volume (BV), fat free dry bone weight (FFBW),
bone ash percentage (Ash%), and bone ash concentration
(BC) were measured according to the methods described
by Kim et al. (2004, 2007). Bones were dried at 100°C for
24 h followed by 76 h refluxing in a Soxhlet apparatus
with hexane. The fat-free bones were then dried again in
an oven at 100°C for 24 h and weighed to obtain FFBW.
The fat-free dry bones were subsequently ashed at 600°C
overnight, cooled in a desiccator, and weighed to obtain
bone ash weight. Ash% was calculated by bone ash weight
divided by FFBW. BC was calculated by dividing the
bone ash weight of each bone by its BV.
Dual-Energy X-Ray Absorptiometry At 20 d (6 dpi)
and 26 d (12 dpi), 2 birds per pen (12 birds/treatment/
time point) was euthanized by cervical dislocation for
scanning using a whole-body dual-energy X-ray
absorptiometry (DEXA) scanner (GE Healthcare, Chi-
cago, IL) (Chen et al., 2020; Tompkins et al., 2022a).
The sample birds were positioned chest up on the scan-
ner. After scanning the whole body, the scanning mode
was set for small animals. Then, the region of interest
was set for individual birds. The data of whole-body
bone mineral density (BMD), bone mineral content
(BMC), and bone area were collected.
Micro-Computed Tomography The right femur was
taken from 1 bird per cage (6 bones/treatment/time
point) at 20 d (6 dpi), and 23 d (9 dpi). The soft tissue
was removed completely. The samples were wrapped
with cheesecloth soaked with phosphate buffered saline
to keep the bones from drying. Then, the bones were
stored at 20°C until further analysis. Before the analy-
sis, the samples were completely defrosted. The bone
was held in a low-dense 50-mL tube, and extra cheese-
cloth was used for keeping the sample firmly inside the
tube holder at a vertical orientation. The tube was then
mounted on the scanning stage. A micro-computed
tomography (micro-CT) scanner (Skyscan 1275;
Bruker micro-CT, Belgium) was used for 2-dimensional
image acquisition at 75 kV and 133 mA, 25 mm pixel
size. Two-D images were transferred to CTAn software
(CTAn, Skyscan) for structure construction and quanti-
fication (Chen et al., 2020; Chen and Kim, 2020). Corti-
cal bone and trabecular bone structures were
reconstructed by CTAn software and separated for bone
4 LOPES ET AL.

Table 2. Growth performance in broilers fed with normal or reduced calcium and phosphorus diets supplemented with 0 IU or 3,000 IU
of 25-hydroxycolecalciferol under Eimeria challenge condition.

0−6 dpi 7−12 dpi

Item1 Ca:P 25-OHD FI (kg) BW (kg) BWG (kg) FCR FI (kg) BW (kg) BWG (kg) FCR
Unch NOR 0 IU 0.491 0.871 0.372 1.48 0.335 1.335 0.464 1.53
RED 0 IU 0.471 0.836 0.341 1.54 0.318 1.238 0.402 1.66
NOR 3,000 IU 0.484 0.864 0.370 1.46 0.329 1.322 0.458 1.52
RED 3,000 IU 0.473 0.857 0.366 1.48 0.328 1.327 0.470 1.52
Cha
NOR 0 IU 0.483 0.573 0.078 2.35 0.195 0.774 0.202 3.98
RED 0 IU 0.476 0.578 0.085 2.49 0.206 0.809 0.230 3.41
NOR 3,000 IU 0.484 0.569 0.075 2.33 0.223 0.804 0.235 2.92
RED 3,000 IU 0.477 0.535 0.043 2.57 0.236 0.723 0.188 3.53
Unch 0.529a 0.856a 0.362a 1.48a 0.345a 1.305a 0.448a 1.55a
Cha 0.405b 0.563b 0.070b 2.43b 0.320b 0.777b 0.213b 3.46b
Ca:P NOR 0.472a 0.718 0.223 1.90 0.337 1.058 0.339 2.52
Ca:P RED 0.462b 0.701 0.208 2.02 0.329 1.024 0.322 2.48
0 IU 0.466 0.714 0.218 1.97 0.326 1.038 0.337 2.64
3,000 IU 0.468 0.706 0.213 1.92 0.339 1.044 0.324 2.37
SEM 0.002 0.01 0.01 0.03 0.006 0.02 0.02 0.25
P value Cha <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
Ca:P 0.033 0.105 0.160 0.060 0.260 0.252 0.423 0.866
25-OHD 0.792 0.443 0.598 0.212 0.055 0.866 0.532 0.283
Cha £ Ca:P 0.779 0.731 0.781 0.296 0.841 0.702 0.710 0.924
Cha £25-OHD 0.446 0.148 0.108 0.779 0.606 0.272 0.412 0.449
25-OHD £ Ca:P 0.083 0.797 0.756 0.581 0.083 0.916 0.985 0.305
Cha £ 25-OHD £ Ca:P 0.418 0.119 0.124 0.232 0.486 0.074 0.081 0.202
Means followed by superscript letters are different by Tukey’s test (P < 0.05) within the column.
a,b
1
Unch: nonchallenge control. Cha: 25,000 E. maxima, 25,000 E. tenella, and 125,000 E. acervulina; Ca:P: mineral levels; NOR: 0.84% of calcium. 0.44%
of phosphorus; RED: 0.64% of calcium. 0.24% of phosphorus; 25-OHD: 25-hydroxycholecalciferol levels; 0 IU: No 25-OHD supplementation; 3,000: 3,000
IU of 25-dyhidroxycholecalciferol/kg of feed; FI: feed intake (kg); BW: body weight (kg); BWG: body weight gain; FCR: feed conversion ratio; dpi: days
postinoculation.

analyses, respectively. The following parameters were
quantified: Tissue volume (TV), Bone volume (BV),
Total bone volume percentage (BV/TV), Trabecular
thickness (Tb.th), Trabecular spacing (Tb.Sp), Tra-
becular number (Tb.N), Trabecular patterns factor
(Tb.pf), Trabecular connectivity (Tb.C), Number of
closed pores (Po.N(cl)), Volume of closed pores (Po.V
(cl)), Surface of closed pores (Po.S(cl)), Closed pores
porosity (Po(cl)), Volume of open pore space (Po.V
(op)), Open pores porosity (Po(op)), Total volume
open pores space (Po.V(tot)), Total porosity (Po
(tot)).
Statistical Analysis
Data were first tested for homogeneity of variances
and normality of studentized residuals. All data were
subjected to 3-way ANOVA, obtaining results for each
factor (25-OHD levels, minerals levels, and challenge) as
well as their interaction. In case of significant differen-
ces, the treatments were compared by Tukey’s test. All
statistical procedures were performed using R software
v. 4.1.1 (R Core Team, 2022). Statements of significance
were based on P < 0.05.
RESULTS
Growth Performance
During the prechallenge period (1−14 d), the BWG,
FI, and FCR did not differ among the treatments (P >
0.05) (data not shown). During the acute challenge
phase (0−6 dpi), challenged birds had inferior FI and
BWG and worse FCR (P < 0.001) when compared with
the nonchallenged birds (Table 2). Moreover, the FI (P
< 0.05) was improved in the RED group when compared
with the NOR group. On recovery phase (7−12 dpi),
whereas the FI, BWG, and FCR were impaired for birds
challenged with Eimeria compared with the unchal-
lenged groups (P < 0.001).
Total Tract Retention of Macrominerals
Effects of Eimeria challenge, and mineral levels were
observed on Ca and P retention at 6 dpi (Table 3). The
challenged birds showed reduced retention of Ca and P
compared with the unchallenged groups (P < 0.001).
Birds from the NOR group showed lower phosphorus
retention than the ones from the RED group
(P = 0.039). In terms of interaction, challenged and
unchallenged birds fed 3,000 IU of 25-OHD showed an
improvement on P retention compared to birds fed with-
out 25-OHD supplementation (P = 0.019) (Table 4).
Intestinal Histomorphometry
At 6 dpi, the villi height in the duodenum and jeju-
num was lower in the challenged birds than the unchal-
lenged birds (P < 0.05) (Supplementary Table 1), but
no differences were visualized in the ileum (P > 0.05).
The crypt depth was greater in the challenged birds
than the unchallenged ones in all 3 segments (P <
0.001). Besides that, the villi: crypt ratios were lower in
 25-HYDROXYCHOLECALCIFEROL IN BROILERS UNDER COCCIDIOSIS 5
Table 3. Total tract retention of calcium and phosphorus in 25-OHD/kg of feed showed higher BAW, Ash%, but
broilers fed with normal or reduced calcium and phosphorus diets lower BC when compared with the groups that received
supplemented with 0 IU or 3,000 IU of 25-hydroxycolecalciferol at diets without 25-OHD supplementation.
6 days postinoculation.
Moreover, an interaction between challenge, mineral
Item1 Ca:P 25-OHD Ca (%) P (%) level, and 25-OHD supplementation was found for BAW
Unch NOR 0 IU 42.81 14.77 (P < 0.05) (Table 6). Within the NOR challenged birds,
RED 0 IU 39.30 42.64 the group supplemented with 3,000 IU of 25-OHD showed
NOR 3,000 IU 42.68 39.36 higher BAW compared with the nonsupplemented birds.
RED 3,000 IU 60.63 59.56
Cha NOR 0 IU 3.03 77.42 No effects of 25-OHD supplementation on BAW were
RED 0 IU 7.49 38.23 identified between NOR unchallenged birds or between
NOR 3,000 IU 16.76 12.65 RED challenged birds that received 0 or 3,000 IU of 25-
RED 3,000 IU 55.01 28.16
Unch 46.35a 39.08a OHD. However, the RED unchallenged birds that received
Cha 15.31b 25.04b 3,000 IU of 25-OHD showed higher BAW compared with
NOR 24.80 8.98b the RED unchallenged nonsupplemented group.
RED 36.86 23.03a
0 IU 17.90 14.56 Comparing the NOR group without 25-OHD supple-
3,000 IU 43.77 28.61 mentation, the challenged groups showed lower BAW
SEM 5.04 6.90 compared to the unchallenged group. However, when
P value Cha <0.001 <0.001
25-OHD 0.083 0.078 receiving 3,000 IU of 25-OHD, NOR birds from the chal-
Ca:P 0.531 0.039 lenged and unchallenged groups showed similar BAW.
Cha £ 25-OHD 0.087 0.019* For RED birds in both 25-OHD supplementation levels,
Cha £ Ca:P 0.519 0.650
25-OHD £ Ca:P 0.592 0.241 the challenged groups showed lower BAW compared to
Cha £ 25-OHD £ Ca:P 0.711 0.956 the unchallenged birds.
a,b
Means followed by superscript letters are different by Tukey’s test (P Finally, within the challenge groups without 25-OHD
< 0.05) within the column. supplementation, no differences were observed for BAW
1
Unch: nonchallenge control; Cha: 25,000 E. maxima, 25,000 E. tenella, between the NOR and RED groups. On the other hand,
and 125,000 E. acervulina; Ca:P: mineral levels; NOR: 0.84% of calcium.
0.44% of phosphorus; RED: 0.64% of calcium. 0.24% of phosphorus; 25- the NOR unchallenged birds without 25-OHD supple-
OHD: 25-hydroxycholecalciferol levels; 0 IU: No 25-OHD supplementa- mentation showed increased BAW compared to RED
tion; 3,000: 3,000 IU of 25-dyhidroxycholecalciferol/kg of feed; Ca: cal- unchallenged birds. Moreover, within the challenge
cium retention (%); P: phosphorus retention (%).
*
Significant interaction. groups with 3,000 IU of 25-OHD, the NOR group
showed higher BAW compared to the RED group,
whereas, when unchallenged and fed 3,000 IU of 25-
the unchallenged groups compared to the unchallenged OHD both groups, the NOR and RED groups showed
group (P < 0.001). similar BAW result.

Oocyst Shedding
At 6 dpi, the effect of Eimeria challenge was observed
(P < 0.001) (Supplementary Table 2). The challenge
groups showed significantly higher oocyst counts for E.
maxima, E. tenella, and E. acervulina compared to the
unchallenged groups.
Bone Ash
Effects of challenge (P < 0.001) were observed for BV,
FFBW, BAW, and BC, while effects of the mineral lev-
els were identified for FFBW (P < 0.05), BAW, Ash%,
and BC (P < 0.001) on 6 dpi (Table 5). The unchal-
lenged birds showed higher BV, FFBW, BAW, and BC
when compared to challenged birds. Birds from the
RED showed lower FFBW, BAW, Ash%, and BC com-
pared to the NOR groups.
At 9 dpi, the challenged groups showed lower BV,
FFBW, and BC when compared to the unchallenged
group (P < 0.001) (Table 5). Furthermore, birds from
the RED groups showed lower FFBW, Ash% (P < 0.05),
and BC (P < 0.001) when compared to the NOR groups.
At this point, effects of 25-OHD (P < 0.05) were
observed on BC, and Ash%. The birds fed 3,000 IU of
DEXA Scan
At 20 d (6 dpi), the whole-body BMD, BMC, and
bone area were influenced by challenge and Ca and P
levels (P < 0.001) (Supplementary Table 3). In chal-
lenged birds, lower BMD was observed when compared
to unchallenged birds. In terms of Ca and P levels, the
NOR groups had higher BMD when compared with the
RED groups. For BMC, the challenged birds, and the
RED group showed lower BMC compared to unchal-
lenged and birds from the NOR, respectively. Moreover,
the groups supplemented with 3,000 IU of 25-OHD/kg
showed higher BMC compared to birds fed diets without
25-OHD supplementation (P < 0.05). At this age, lower
bone area was observed in challenged birds and the
RED group compared to unchallenged birds and the
NOR group, respectively.
At 26 d (12 dpi), the BMD (P = 0.006), BMC and
bone area (P < 0.001) were influenced by challenge,
while BMC and bone area were influenced by the min-
eral levels (P < 0.001). The birds from the challenged
group showed lower BMD, BMC and bone area, and
birds from the RED group showed lower BMC and bone
area compared to the NOR group. At this age, 25-OHD
supplementation showed effects on BMC (P < 0.05).
6 LOPES ET AL.

Table 4. Interaction between challenge and 25-hydroxycholecal- on 6 dpi. At the same sampling point, the Eimeria spp.
ciferol supplementation on total tract retention of phosphorus in challenge birds showed lower total BV (P < 0.05) and
broilers challenged with mixed Eimeria spp. at 6 days postinocu- BV/TV (P < 0.001), whereas decreased BV/TV was
lation.
observed in the RED group (P < 0.05).
Item1 25-OHD P (%) At 6 dpi, the 25-OHD supplementation increased the
Unch trabecular TV (P < 0.05) when compared to the nonsup-
0 IU 28.70b plemented group, whereas the coccidiosis challenge
3,000 IU 49.46a decreased TV (P < 0.05), Tb.th (P < 0.05), and Tb.pf
Cha
0 IU 57.83b (P < 0.001) compared to the unchallenged birds. The
3,000 IU 7.75a birds fed unbalanced and reduced mineral level (RED)
SEM 6.90 showed an increase on Tb.pf (P < 0.05) compared to the
P value 0.019
balanced and normalized mineral level group (NOR)
a,b
Means followed by superscript letters are different by Tukey’s test (P (Table 7). An interaction was observed between 25-
< 0.05) within the column.
1
Unch: nonchallenge control; Cha: 25,000 E. maxima, 25,000 E. tenella, OHD and Eimeria spp. challenge for BV and Tb.C
and 125,000 E. acervulina; 25-OHD: 25-hydroxycholecalciferol levels; 0 (Table 8). No differences were found between the chal-
IU: No 25-OHD supplementation; 3,000: 3,000 IU of 25-dyhidroxychole- lenge groups regardless of 25-OHD supplementation on
calciferol/kg of feed; Ca: calcium retention (%); P: phosphorus retention
(%). BV and Tb.C, whereas the 25-OHD effects were
observed in the unchallenged groups with the 3,000 IU
25OHD, showing higher BV and Tb.C.
Birds fed 3,000 IU of 25-OHD/kg of feed showed higher For cortical bone parameters at 6 dpi, 25-OHD sup-
BMC when compared to birds fed diets without 25- plementation groups showed an increased TV (P <
OHD supplementation. 0.05), BV (P < 0.05), and Po.N(cl) (P < 0.05) when
compared to the 0 IU supplementation group. The chal-
lenged birds showed a decreased TV (P < 0.05), BV (P
Micro-CT
< 0.05), Po.V(cl) (P < 0.05), Po.S(cl) (P < 0.05), and
Effects of 25-OHD were observed for total bone TV, Po.(cl) (P < 0.05) compared to the unchallenged group,
and BV (P < 0.05) (Supplementary Table 4). Broilers whereas there were no differences in Po.V(op), P(op),
fed 3,000 IU of 25-OHD showed an increase on TV and Po.V(tot) and Po(tot) between all parameters analyzed
BV compared to birds fed diets without 0 IU of 25-OHD and their interactions (Supplementary Table 5).

Table 5. Bone volume, fat-free dry bone weight, ash weight, ash percentage, and bone ash concentration in broilers fed with normal or
reduced calcium and phosphorus diets supplemented with 0 IU or 3,000 IU of 25-hydroxycolecalciferol under Eimeria challenge condition
at 6 and 9 days postinoculation (dpi).

6 dpi 9 dpi
1 3
Item Ca:P 25-OHD BV (cm ) FFBW (g) BAW (g) Ash% BC (g/cm ) BV (cm ) FFBW (g) BAW (g) Ash% BC (g/cm3)
3 3

Unch NOR 0 IU 8.15 4.98 1.84 46.42 0.23 13.22 7.95 3.07 46.21 0.23
RED 0 IU 8.28 4.84 1.65 43.24 0.20 12.32 6.87 2.38 42.09 0.19
NOR 3,000 IU 8.31 4.97 1.93 50.01 0.23 13.25 7.66 3.03 46.91 0.23
RED 3,000 IU 8.35 4.97 1.73 44.04 0.21 12.90 7.47 2.80 45.80 0.22
Cha
NOR 0 IU 7.77 4.63 1.61 45.91 0.21 10.23 5.99 2.22 45.53 0.22
RED 0 IU 7.28 4.24 1.41 44.00 0.19 9.77 5.51 1.89 42.34 0.19
NOR 3,000 IU 7.71 4.57 1.62 45.82 0.21 10.40 6.18 2.59 50.45 0.25
RED 3,000 IU 6.95 4.18 1.39 44.46 0.20 10.33 5.92 2.09 43.18 0.20
Unch 8.28a 4.94a 1.79a 45.93 0.22a 12.92a 7.49a 2.82 45.25 0.22
Cha 7.43b 4.40b 1.51b 45.05 0.20b 10.18b 5.90b 2.20 45.37 0.22
Ca:P NOR 7.99 4.79a 1.75a 47.04a 0.22a 11.78 6.95a 2.73 47.28a 0.23a
Ca:P RED 7.72 4.56b 1.54b 43.93b 0.20b 11.33 6.44b 2.29 43.35b 0.20b
0 IU 7.87 4.67 1.63 44.89 0.21 11.38 6.58 2.39 44.04b 0.21a
3,000 IU 7.43 4.67 1.67 46.08 0.21 11.72 6.81 2.63 46.58a 0.11b
SEM 0.17 0.08 0.04 0.78 0.004 0.17 0.17 0.07 1.24 0.006
P value Cha <0.001 <0.001 <0.001 0.591 0.003 <0.001 <0.001 <0.001 0.925 0.180
Ca:P 0.136 0.010 <0.001 <0.001 <0.001 0.082 0.005 <0.001 0.003 <0.001
25-OHD 0.834 0.976 0.317 0.359 0.134 0.185 0.195 0.002 0.048 0.012
Cha £ Ca:P 0.053 0.067 0.800 0.066 0.105 0.478 0.438 0.757 0.301 0.265
Cha £ 25-OHD 0.391 0.476 0.256 0.405 0.762 0.907 0.678 0.524 0.787 0.327
25-OHD £ Ca:P 0.593 0.705 0.825 0.900 0.697 0.349 0.118 0.340 0.830 0.937
Cha £ 25- 0.798 0.690 0.945 0.537 0.728 0.881 0.338 0.040* 0.162 0.196
OHD £ Ca:P
Means followed by superscript letters are different by Tukey’s test (P < 0.05) within the column.
a,b
1
Unch: nonchallenge control; Cha: 25,000 E. maxima, 25,000 E. tenella, and 125,000 E. acervulina; Ca:P: mineral levels; NOR: 0.84% of calcium. 0.44%
of phosphorus; RED: 0.64% of calcium. 0.24% of phosphorus; 25-OHD: 25-hydroxycholecalciferol levels; 0 IU: No 25-OHD supplementation; 3,000: 3,000
IU of 25-dyhidroxycholecalciferol/kg of feed; BV: bone volume (cm3). FFBW: fat-free bone weight (g); BAW: bone ash weight (g); Ash%: ash percentage;
BC: bone ash concentration (g/cm3); dpi: days postinoculation.
*
Significant interaction (P < 0.05).
 25-HYDROXYCHOLECALCIFEROL IN BROILERS UNDER COCCIDIOSIS 7
Table 6. Effect of 25-hydroxycholecalciferol, Eimeria challenge, and challenge for Tb.C (P < 0.05) (Tables 9 and 10).
and Ca and phosphorus levels on tibia bone ash weight in broilers Among the groups receiving 3,000 IU of 25-OHD, there
at 9 days postinoculation. was no difference between challenged and unchallenged
BAW (g) 25-OHD birds, however, within the nonsupplemented birds, the
Item1 Challenge 0 IU 3,000 IU challenged ones had worse Tb.C compared to the
,b,B unchallenged group. No differences were observed in Tb.
NOR Cha 2.22** 2.59**,a,A
Unch 3.07**,a,A 3.03*,a,A th, and Tb.Sp at this point.
Considering cortical bone parameters, the 25-OHD
RED Cha 1.89*,a,B 2.09*,a,B supplementation improved BV (P < 0.05), Po.N(cl) (P
Unch 2.38*,b,A 2.80*,a,A
SEM 0.07 < 0.05), Po.V(cl) (P < 0.05), Po.S(cl) (P < 0.05), and
P value Cha £ 25-OHD £Ca:P 0.040 Po.(cl) (P < 0.05). The Eimeria challenge worsened cor-
a,b
Means followed by superscript letters are different by Tukey’s test (P tical TV, BV, Po.N(cl), Po.S(cl), Po.V(op) (P < 0.001),
< 0.05) within the row for 25-OHD/Cha and Ca:P.
A,B
Po.V(cl) (P = 0.001), and Po.(cl) (P < 0.05) (Table 11).
Means followed by superscript letters are different by Tukey’s test An interaction was observed between 25-OHD and min-
(P < 0.05) within the column for Cha/Ca:P and 25-OHD.
*,**
Means followed by superscript sign are different by Tukey’s test (P eral levels for Po(op) (P < 0.05), and Po(tot) (P < 0.05)
< 0.05) within the column for Ca:P/Cha and 25-OHD. (Tables 11 and 12). When the birds were fed diets sup-
1
Unch: nonchallenge control; Cha: 25,000 E. maxima, 25,000 E. tenella, plemented with 3,000 IU of 25-OHD regardless of the
and 125,000 E. acervulina; Ca:P: mineral levels; NOR: 0.84% of calcium.
0.44% of phosphorus; RED: 0.64% of calcium. 0.24% of phosphorus; 25- mineral balance and level, there were no differences for
OHD: 25-hydroxyhcolecalciferol levels; 0 IU: No 25-OHD supplementa- Po(op) and Po(tot), whereas, in the 25-OHD nonsupple-
tion; 3,000: 3,000 IU of 25-dyhidroxycholecalciferol/kg of feed; BAW: mented group, the unbalanced and reduced mineral
bone ash weight (g).
groups had lower Po(op) and Po(tot).

At 9 dpi, for total femur bone structure parameters, DISCUSSION

the Eimeria challenge depressed TV (P < 0.001) and BV
(P < 0.001), while the RED group reduced BV (P <
0.05) and BV/TV (P < 0.05) (Supplementary Table 6).
For trabecular bone, the avian coccidiosis dropped TV
(P < 0.001) and BV (P < 0.05). The mineral unbalance
and reduction (RED group) showed a decrease on Tb.pf
(P < 0.05). There was an interaction between 25-OHD
In the current study, during 0 to 6 dpi and 7 to 12 dpi,
the Eimeria infection had a negative effect on growth
performance. The growth performance impairment
observed during coccidiosis could be associated with par-
asite-induced anorexia and a deleterious impact on the
intestine brush border structure, which may negatively

Table 7. Effect of 25-hydroxycholecalciferol and calcium and phosphorus levels on femur trabecular bone traits of broilers challenged
with mixed Eimeria spp. at 6 days postinoculation (dpi).

Trabecular Bone (6 dpi)

Item1 Ca:P 25-OHD TV (mm3) BV (mm3) Tb.th (mm) Tb.Sp (mm) Tb.N (1/mm) Tb.pf (1/mm) Tb.C
Unch NOR 0 IU 171.53 12.15 0.124 2.65 0.55 8.94 1655
RED 0 IU 150.75 10.32 0.129 2.16 0.53 9.05 1324
NOR 3,000 IU 185.52 17.56 0.123 2.08 0.75 8.39 2675
RED 3,000 IU 207.51 15.62 0.119 2.28 0.64 10.09 2438
Cha
NOR 0 IU 190.45 13.86 0.121 2.36 0.56 10.30 2140
RED 0 IU 214.56 13.85 0.118 2.39 0.54 10.38 2237
NOR 3,000 IU 192.51 11.42 0.119 2.77 0.49 10.21 1797
RED 3,000 IU 226.83 12.93 0.115 2.68 0.49 11.42 2203
Unch 178.83b 13.91 0.124a 2.29 0.62 9.12a 2023
Cha 206.09a 13.02 0.118b 2.55 0.52 10.58b 2094
Ca:P NOR 185.01 13.75 0.122 2.46 0.59 9.46b 2067
Ca:P RED 199.92 13.18 0.120 2.37 0.55 10.23a 2050
0 IU 181.83b 12.55 0.123 2.39 0.55 9.672 1839
3,000 IU 203.10a 14.38 0.119 2.45 0.59 10.03 2278
SEM 9.41 1.52 0.002 0.18 0.05 0.38 236.00
P value Cha 0.006 0.558 0.033 0.174 0.073 <0.001 0.764
Ca:P 0.121 0.710 0.467 0.639 0.444 0.049 0.945
25-OHD 0.029* 0.233 0.131 0.740 0.409 0.351 0.069
Cha £ Ca:P 0.136 0.392 0.532 0.746 0.630 0.739 0.262
Cha £ 25-OHD 0.141 0.026* 0.568 0.128 0.052 0.758 0.011*
25-OHD £ Ca:P 0.167 0.815 0.389 0.455 0.73 0.083 0.671
Cha £ 25-OHD £ Ca:P 0.392 0.789 0.423 0.287 0.61 0.768 0.821
a,b
Means followed by superscript letters are different by Tukey’s test (P < 0.05) within the column.
1
Unch: nonchallenge control; Cha: 25.000 E. maxima, 25.000 E. tenella, and 125.000 E. acervulina; Ca:P: mineral levels; NOR: 0.84% of calcium. 0.44%
of phosphorus; RED: 0.64% of calcium. 0.24% of phosphorus; 25-OHD: 25-hydroxycholecalciferol levels; 0 IU: No 25-OHD supplementation; 3,000: 3,000
IU of 25-dyhidroxycholecalciferol/kg of feed; TV: tissue volume (mm3); BV: bone volume (mm3); Tb.th: trabecular thickness (mm); Tb.Sp: trabecular
spacing (mm); Tb.N: trabecular number (1/mm); Tb.pf: trabecular pattern factor (1/mm); Tb.C: trabecular connectivity; dpi: days postinoculation.
*
Significant Interaction (P < 0.05).
8 LOPES ET AL.

Table 8. Interaction between mixed Eimeria challenge and 25- difference was not sufficient to influence performance
hydroxycholecalciferol supplementation on trabecular bone vol- results. Shi et al. (2022) observed that phytase supple-
ume and trabecular connectivity at 6 days postinoculation (dpi). mentation improved FI for broilers fed with 0.75% of Ca
Trabecular bone (6 dpi) and 0.23% of available P due to mineral retention and
Item1 25-OHD BV (mm3) Tb.C total P percentage enhancement. Other studies showed
a positive effect of dietary vitamin d supplementation on
Unch
0 IU 11.2b 1490b FI, and an increase in the gene expression of small intes-
3,000 IU 16.6’ 2557a tine calcium and phosphorus transporters during the
Cha growing phase (Wu et al., 2022). Therefore, it is possible
0 IU 13.9’ 2189’
3,000 IU 12.2’ 2000’ that dietary supplemented VitD can support broiler per-
SEM 1.52 236.00 formance by increasing mineral absorption. However,
P value 0.026 0.011 the intensity of the Eimeria challenge together with the
a,b
Means followed by superscript letters are different by Tukey’s test (P mid-severe dietary mineral reduction used in the present
< 0.05) within the column. experiment could have been too intense and masked pos-
1
Unch: nonchallenge control; Cha: 25,000 E. maxima, 25,000 E. tenella,
and 125,000 E. acervulina; Ca:P: 25-OHD: 25-hydroxycholecalciferol lev- sible 25-OHD effects.
els; 0 IU: No 25-OHD supplementation; 3,000: 3,000 IU of 25-dyhidroxy- The lack of interaction between 25-OHD and mineral
cholecalciferol/kg of feed; BV: bone volume (mm3). Tb.C: trabecular level on performance observed in this study could also
connectivity. dpi: days postinoculation.
have occurred due to the discrepancy between the 25-
OHD level used (3,000 vs. 5,000 IU recommended by the
affect the nutrient digestion and absorption due to para- breeder manual) and the intense phosphorus reduction
site replication (Adams et al., 1996; Teng et al., 2020a; (47.61%) that probably require a higher 25-OHD inclu-
Abdelhady et al., 2021; Sharma et al., 2022; Liu et al., sion level. Still, further studies evaluating the effects of
2023). Eimeria damages in the intestine structure have 25-OHD in levels higher than the one used here are nec-
extensive and profound negative changes in other sys- essary to confirm the hypothesis that vitamin D is able
tems, such as bone. to mitigate the coccidiosis and dietary mineral reduction
Regarding performance, the RED group showed a detrimental effects on broilers’ performance.
decrease in FI by 3.0%. Besides the birds from the RED Broilers fed reduced levels of Ca and P can adapt to
group supplemented with 3,000 UI of 25-OHD presented mineral deficiency due to a compensatory increase of
a numerically increased phosphorus retention compared serum VitD that by upregulating the expression of VDR
to the nonsupplemented birds (P = 0.241), this transporters ion the small intestine, leading to a higher

Table 9. Effect of 25-hydroxycholecalciferol supplementation and calcium and phosphorus levels on femur trabecular bone traits of
broilers challenged with mixed Eimeria spp. at 9 days postinoculation (dpi).

Trabecular bone (9 dpi)

Item1 Ca:P 25-OHD TV (mm3) BV (mm3) Tb.th (mm) Tb.Sp (mm) Tb.N (1/mm) Tb.pf (1/mm) Tb.C
Unch NOR 0 IU 314 35.4 0.133 1.63 0.83 6.46 4,075
RED 0 IU 322 28.5 0.140 2.36 0.64 6.90 3,445
NOR 3,000 IU 285 27.5 0.144 2.57 0.67 5.34 2,616
RED 3,000 IU 301 26.8 0.132 2.49 0.67 7.14 3,547
Cha
NOR 0 IU 262 23.7 0.145 2.49 0.61 5.99 2,283
RED 0 IU 243 18.1 0.146 2.67 0.50 7.61 2,000
NOR 3,000 IU 239 24.9 0.142 2.28 0.75 5.98 2,875
RED 3,000 IU 279 25.0 0.134 2.35 0.66 7.43 3,127
Unch 314 35.4a 0.133 1.63a 0.83 6.46 4,075a
Cha 306 29.5b 0.137 2.26b 0.70 6.46 3,421b
Ca:P NOR 256 22.9 0.142 2.45 0.63 6.75a 2,571
Ca:P RED 275 27.9 0.141 2.24 0.72 5.94b 2,962
0 IU 286 24.4 0.138 2.47 0.62 7.26 3,030
3,000 IU 276.6 26.08 0.141 2.42 0.690 6.47 3,041
SEM 11.58 2.51 0.003 0.23 0.05 0.40 194.44
P value Cha <0.001 0.011 0.199 0.429 0.228 0.478 0.026
Ca:P 0.335 0.201 0.441 0.333 0.099 0.002 0.855
25-OHD 0.428 0.880 0.390 0.571 0.480 0.518 0.808
Cha £ Ca:P 0.966 0.845 0.873 0.674 0.987 0.605 0.822
Cha £ VitD 0.188 0.084 0.221 0.093 0.073 0.675 0.043*
VitD £ Ca:P 0.145 0.248 0.066 0.328 0.364 0.466 0.162
Cha £ VitD £ Ca:P 0.270 0.962 0.539 0.447 0.451 0.352 0.490
Means followed by superscript letters are different by Tukey’s test (P < 0.05) within the column.
a,b
1
Unch: nonchallenge control; Cha: 25,000 E. maxima, 25,000 E. tenella, and 125,000 E. acervulina; Ca:P: mineral levels; NOR: 0.84% of calcium. 0.44%
of phosphorus; RED: 0.64% of calcium. 0.24% of phosphorus; 25-OHD: 25-hydroxycholecalciferol levels; 0 IU: No 25-OHD supplementation; 3,000: 3,000
IU of 25-dyhidroxycholecalciferol/kg of feed; TV: tissue volume (mm3); BV: bone volume (mm3); Tb.th: trabecular thickness (mm); Tb.Sp: trabecular
spacing (mm); Tb.N: trabecular number (1/mm); Tb.pf: trabecular pattern factor (1/mm); Tb.C: trabecular connectivity; dpi: days postinoculation.
*
Significant interaction (P < 0.05).
 25-HYDROXYCHOLECALCIFEROL IN BROILERS UNDER COCCIDIOSIS 9
Table 10. Interaction between mixed Eimeria challenge and 25- In addition, P retention was improved when chal-
hydroxycholecalciferol supplementation levels on trabecular con- lenged birds were fed 3,000 IU of 25-OHD, indicating
nectivity at 9 days postinoculation (dpi). that the 25-OHD supplementation might be advanta-
Trabecular Bone (9 dpi) geous for mineral utilization during coccidiosis. The
Item1 25-OHD Tb.C phosphorus is transported by sodium-phosphate
0 IU cotransporters (NaPi-IIb) of enterocytes present in the
Unch 3,761a duodenum, jejunum, and ileum (Han et al., 2018b). In
Cha 2,142b broilers, vitamin D in its active form can regulate circu-
3,000 IU
Unch 3,082a lating calcium and phosphorus levels by increasing
Cha 3,001a CaBPD-28k, PMCA1b, and NCX1 in the small intestine
SEM 194.44 (Wu et al., 2022) and the renal NaPi-IIa, and NaPi-IIc
P value 0.043
a,b
activity (Han et al., 2018a).
Means followed by superscript letters are different by Tukey’s test (P
< 0.05) within the column.
There is conflicting evidence about the effects of VitD
1
Unch: nonchallenge control; Cha: 25,000 E. maxima, 25,000 E. tenella, on Eimeria oocyst shedding. Sakkas et al. (2018) found
and 125,000 E. acervulina; Ca:P: 25-OHD: 25-hydroxycholecalciferol lev- that 4,000 IU of VitD increased the oocyst shedding of
els; 0 IU: No 25-OHD supplementation; 3,000: 3,000 IU 25-dyhidroxycho-
lecalciferol/kg of feed; Tb.C: trabecular connectivity; dpi: days
broilers infected a low E. maxima infection (7,000
postinoculation. oocysts/mL), while Shanmugasundaram et al. (2019)
observed that 25-OHD supplementation decreased the
oocyst shedding in turkeys infected with a medium E.
maxima infection (20,000 occysts/mL). In the present
ability to absorb Ca and P (Yan et al., 2005). Our results study, birds were infected with a considered mid-high
show that birds fed reduced levels of Ca and P showed infection level (Teng et al., 2020a), and the oocyst shed-
an increase on P and Ca retention. This result agrees ding was not influenced by Ca and P balance and levels
with Shi et al. (2022) who reported that the chicks fed or 25-OHD supplementation. It is important to highlight
low Ca and P diet increased Ca and P retention. More- that the current and previous studies have differences in
over, the group with unbalanced and reduced dietary Ca the parasite species used for challenge (mixed E. acervu-
and P and fed with 3,000 IU of 25-OHD increased the lina, E. maxima, E. tenella vs. E. maxima only), infec-
Ca retention by 72.5% compared to the nonsupple- tion intensity (mid-high vs. low vs. mid) levels of dietary
mented group. 25-OHD (3,000 IU vs. 4,000 IU vs. 4,400 IU), and bird

Table 11. Effect of 25-hydroxycholecalciferol supplementation and calcium and phosphorus levels on femur cortical bone traits of
broilers challenged with mixed Eimeria spp. at 9 days postinoculation (9 dpi).

Cortical Bone (9 dpi)

BV Po.V (cl) Po.S (cl) Po(cl) Po.V (op) Po.V (tot) Po (tot)
Item1 Ca:P 25-OHD TV (mm3) (mm3) Po.N (cl) (mm3) (mm2) (%) (mm3) Po (op) (%) (mm3) (%)
Unch NOR 0 IU 230.54 165.61 703.16 0.60 32.80 0.34 64.33 28.26 64.93 28.51
RED 0 IU 194.13 145.34 846.66 0.60 35.06 0.38 48.18 23.93 48.79 24.22
NOR 3,000 IU 227.16 174.12 875.16 0.99 50.49 0.55 52.04 22.68 53.04 23.12
RED 3,000 IU 225.46 168.42 1137.5 0.80 47.25 0.47 56.22 25.08 57.03 25.43
Cha
NOR 0 IU 165.38 125.68 520.6 0.53 27.72 0.39 39.16 23.44 39.70 23.74
RED 0 IU 123.95 100.16 362.3 0.23 13.67 0.21 23.55 17.48 23.78 17.66
NOR 3,000 IU 186.51 139.71 620.1 0.61 31.95 0.43 46.18 24.15 46.80 24.48
RED 3,000 IU 162.64 119.44 631.3 0.41 25.03 0.32 42.77 25.08 43.19 25.32
Unch 219.32a 163.37a 890.6a 0.75a 41.40a 0.44a 55.19a 24.99 55.95a 25.32
Cha 159.62b 121.25b 533.6b 0.45b 24.59b 0.34b 37.92b 22.54 38.37b 22.80
Ca:P NOR 202.40a 151.28a 679.7 0.69 35.74 0.43 50.43 24.63 51.12 24.96
Ca:P RED 176.54b 133.34b 744.4 0.51 30.25 0.35 42.68 22.89 43.20 23.16
0 IU 200.45 134.20b 608.2b 0.49b 27.32b 0.34b 49.30 24.25 50.01 24.59
3,000 IU 178.50 150.43a 816.0a 0.71a 38.68a 0.45a 43.81 23.28 44.30 23.53
SEM 11.02 7.66 93.45 0.08 4.62 0.04 4.26 1.58 4.29 1.57
P value Cha <0.001 <0.001 <0.001 0.001 <0.001 0.022 <0.001 0.130 <0.001 0.117
Ca:P 0.023 0.024 0.492 0.052 0.241 0.053 0.076 0.279 0.072 0.260
25-OHD 0.053 0.040 0.031 0.016 0.018 0.009 0.204 0.544 0.190 0.507
Cha £ Ca:P 0.540 0.521 0.146 0.372 0.285 0.132 0.680 0.630 0.669 0.607
Cha £ VitD 0.473 0.955 0.802 0.346 0.444 0.343 0.081 0.051 0.086 0.052
VitD £ Ca:P 0.242 0.521 0.445 0.809 0.930 0.699 0.063 0.037* 0.066 0.038*
Cha £ VitD £ Ca:P 0.699 0.762 0.892 0.405 0.497 0.249 0.636 0.979 0.651 0.960
a,b
Means followed by superscript letters are different by Tukey’s test (P < 0.05) within the column.
1
Unch: nonchallenge control; Cha: 25,000 E. maxima, 25,000 E. tenella, and 125,000 E. acervulina; Ca:P: mineral levels; NOR: 0.84% of calcium. 0.44%
of phosphorus; RED: 0.64% of calcium. 0.24% of phosphorus; 25-OHD: 25-hydroxycholecalciferol levels; 0 IU: No 25-OHD supplementation; 3,000: 3,000
IU of 25-dyhidroxycholecalciferol/kg of feed; TV: tissue volume (mm3); BV: bone volume (mm3); Po.N(cl): number of closed pores; Po.V(cl): volume of
closed pores (mm3); Po.S(cl): surface of closed pores (mm2); Po(cl): closed pores porosity (%); Po.V(op): volume open pore space (mm3); Po(op): open
pores porosity (%); Po.V(tot): total volume open pores space (mm3); Po(tot): total porosity (%); dpi: days postinoculation.
*
Significant interaction (P < 0.05).
10 LOPES ET AL.
Table 12. Interaction between mixed Eimeria challenge and 25-
Hydroxycholecalciferol supplementation on cortical open pores
porosity and cortical total porosity at 9 days postinoculation
(dpi).
Cortical Bone (9 dpi)
Item1 Ca:P Po(op) (%) Po(tot) (%)
0 IU
Nor 25.9a 26.1a
Red 20.7b 20.9b
3,000 IU
Nor 25.1a 25.4a
Red 23.4a 23.8a
SEM 1.58 1.57
P value 0.037 0.038
a,b
Means followed by superscript letters are different by Tukey’s test (P
< 0.05) within the column.
1
Ca;P: mineral levels; NOR: 0.84% of calcium. 0.44% of phosphorus;
RED: 0.64% of calcium. 0.24% of phosphorus; 0 IU: No 25-dyhidroxycho-
lecalciferol supplementation; 3,000: 3,000 IU of 25-dyhidroxycholecalci-
ferol/kg of feed; Po(op): open pores porosity (%); Po(tot): total porosity
(%); dpi: days postinoculation.
species (broilers vs. turkeys), which could lead to con-
trasting results.
Eimeria challenge, dietary Ca and P reduction, and
VitD supplementation level can influence broiler bone
quality (Kim et al., 2011; Kakhiki et al., 2019; Sakkas
et al., 2019; Zhang et al., 2020; Shi et al., 2022; Tomp-
kins et al., 2023). Regarding bone quality, bone ash
weight is an important bone health indicator in
broilers, because it can be straightly related to bone
mineral content and bone breaking strength and it has
been linked to a better degree of bone mineralization
(Kim et al., 2012; Shao et al., 2019). In the current
study, it was possible to observe that independently of
mineral levels, the 25-OHD supplementation at the
level of 3,000 IU/kg of feed was capable of counteract
the Eimeria negative effect on BAW. In the absence
of challenge, the 25-OHD supplementation improved
BAW in the RED group. However, it should be noted
that when providing a standardized and balanced Ca
and P diet in absence of challenge, the 3,000 IU of 25-
OHD supplementation did not show effect on BAW.
This result was expected, because birds from this
group have not experienced any adverse situation that
required an activation of the intestinal calcium and
phosphorus absorption pathway via VitD to increase
circulating Ca and P levels and ensure a sufficient Ca
and P supply for bone turnover balance.
Furthermore, on 9 dpi, the groups fed with 3,000 IU
of 25-OHD showed increase %Ash by 5.45%. The bone
ash measurement is recognized for its sensitivity to fluc-
tuations in the dietary phosphorus levels, which can
explain the results observed for BAW and %Ash in the
current experiment. Based on the bone ash results here
presented, although the effects of 25-OHD were not
observed during the peak of infection (6 dpi), the 25-
OHD supplementation stimulated bone growth and
mineralization through the recovery phase, indicating
that 25-OHD supplementation is critical to compensate
for the bone loss caused by low Ca & P diet and coccidi-
osis.
The Micro-CT scanner is a high-resolution tomogra-
phy used to visualize and measure cortical, trabecular,
and total bone morphological parameters in a nonde-
structive way and has been used to evaluate bone health
in animal models such as rodents (Wu et al., 2015; Ning
et al., 2022), laying hens (Castro et al., 2020; Chen et
al., 2020; Chen and Kim, 2020; White et al., 2023),
quails (Lee et al., 2023), and broilers (Yair et al., 2012;
Tompkins et al., 2022a,b, 2023). However, to our knowl-
edge, there are still no published papers evaluating the
effects of 25-OHD supplementation and dietary mineral
balance and level on bone microarchitecture parameters
in broilers during Eimeria spp. infection. In this study,
the 3-D bone analyses provided by micro-CT were used
to evaluate microstructural traits of metaphyseal bone
to understand the morphological changes occurring dur-
ing the peak and the recovery phase of a coccidiosis chal-
lenge in broilers fed the reduced Ca & P diets
supplemented with 25-OHD, and how these factors
interact in an infectious situation.
Based on the current results, the detrimental effects
by Eimeria infection were observed in the cancellous
and compact bone and extended until the recovery
phase. Possibly, during coccidiosis, birds are under poor
bone growth and mineralization due to the duodenal
and jejunal tissue damage caused by Eimeria spp. that
makes the availability of Ca and P insufficient for
hydroxyapatite deposition. During the recovery phase,
the lack of effect of coccidiosis on total bone BV/TV at
9 dpi indicates the start of the positive balance between
bone production and bone resorption. In addition,
because long bone growth is closely correlated with
weight gain and body weight (Applegate and Lilburn,
2002), quite possibly, challenged birds with performance
delay may still have lag behind their optimum skeletal
development for muscle support. As a result, there is an
increased risk of overload on the skeletal system, increas-
ing the chances of bone pathologies, and decreasing wel-
fare and productivity.
In the trabecular bone, Eimeria spp. infection wors-
ened bone quality by reducing bone strength. That can
be noted by the lower trabecular thickness, connectivity,
and pattern in the challenged group. Coccidiosis can
lead to increased gene expression of the receptor activa-
tor of nuclear factor kappa-b ligand (RANK-L) pro-
tein, which is responsible for the differentiation and
activation of osteoclasts (Oikeh et al., 2019; Sharma et
al., 2023). During normal bone remodeling an increase in
deep bone resorption occurs after osteoclasts activation,
but in pathological situations the excessive RANK-L
production leads to a structural change (Boyle et al.,
2003) as observed in the current study. The reduced
number of trabeculae as well as thin, fragmented, poorly
mineralized, and misaligned bone trabeculae indicate
less mineral deposition, more bone resorption, or both
occurring simultaneously, and are consistent with the
development of osteopenic metabolic bone diseases such
as fibrous osteodystrophy (Pashuck et al., 2009).
Whereas in the cortical bone, the smaller closed pore
volume and surface area indicate a delayed development
 25-HYDROXYCHOLECALCIFEROL IN BROILERS UNDER COCCIDIOSIS
of the Haversian system, since it corresponds to primary
or secondary osteons that exists during early trabecula-
rization stages (Chappard et al., 2013). Moreover, the
higher open pore number can be related to cortical bone
loss (Perilli et al., 2015; Chen et al., 2020) that leads to
decrease due to severe bone resorption by osteoclasts.
The present results show that this lower cortical bone
quality lasted until the recovery phase. Therefore, a
medium-high Eimeria infection can decrease broiler
bone quality profoundly and extensively due to its acute
and medium-term infection.
Another endanger caused by coccidiosis is the bal-
ance redox disruption, increased liver lipid peroxida-
tion, and ROS production that lead to oxidative stress
(Tompkins et al., 2022a). During oxidative stress there
is an increase in the production of enzymes that induces
bone marrow mesenchymal stem cells toward adipo-
genic differentiation instead of bone cells lineage
(Tompkins et al., 2022a), suppress the expression of
genes correlated to osteoblast activity, osteoclasts mat-
uration and activity (Tompkins et al., 2022b, 2023).
The activation of adipogenic differentiation cascade
worsens bone structural traits that can be seen by an
increased bone mineral loss, and poor bone growth
rate. In the current study, trabecular connectivity
results indicates that 3,000 IU of 25-OHD can increase
trabecular bone mineralization and connectivity in the
absence of challenge, but these levels cannot counter
act Eimeria spp. effects during the peak of the infec-
tion. After the peak, the 25-OHD was able to guarantee
trabecular connectivity on challenged birds. Besides
the 25-OHD direct effect on bone development, a
decrease on superoxide dismutase in LPS-challenged
broilers supplemented with VitD was previously
observed (Zhang et al., 2022) and could indicate that
vitamin D has a role on oxidative metabolism. There is
evidences that 25-OHD can modulate bone remodeling
during oxidative stress by decreasing the RANKL and
increasing osteoprotegerin gene expression (Zhang et
al., 2021; Choppa and Kim, 2023), playing a role in
increasing osteoblasts vitamin D receptor gene expres-
sion (Nakamichi et al., 2017), insulin like-growth factor
(Krohn et al., 2003) in mice’s chondroblasts, and
growth factor 23 (FGF-23) in humans (Fernandez-
Cancio et al., 2009).
In conclusion, a mixed mid-high Eimeria challenge
impaired bone quality by both, direct and indirectly
ways. The dietary supplementation of 25-OHD
improved mineral retention, and bone quality during
Eimeria challenge. However, the dosage of 3,000 IU of
25-OHD was not able to counteract the bone quality
negative effects of coccidiosis and mid-severe dietary
mineral reduction when together. Further investigation
is needed to evaluate whether the increase of 25-OHD
dietary level higher than 3,000 IU is capable to counter-
act the detrimental effects of feed mineral reduction on
bone quality parameters during mid-high Eimeria chal-
lenge. Further studies are necessary to understand the
role of dietary 25-OHD supplementation on bone resorp-
tion by osteoclasts during coccidiosis.
11
ACKNOWLEDGMENTS
This work was financed in part by the Coordenaç~
ao de
Aperfeiçoamento de Pessoal de Nível Superior − Brazil
(CAPES) − Finance Code 001.
DISCLOSURES
There is no conflict of interest.
SUPPLEMENTARY MATERIALS
Supplementary material associated with this article
can be found in the online version at doi:10.1016/j.
psj.2023.103267.
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