animals

Article
Dietary 25-Hydroxycholecalciferol Supplementation as a
Vitamin D3 Substitute Improves Performance, Egg Quality,
Blood Indexes, Jejunal Morphology, and Tibia Quality in
Late-Phase Laying Hens
Shan Gao 1 , Kai Qiu 1 , Junjie Zheng 2 , Haijun Zhang 1 , Jing Wang 1 , Xiaolong Qi 3, * and Shugeng Wu 1, *

1 Laboratory of Quality and Safety Risk Assessment for Animal Products on Feed Hazards (Beijing),
the Ministry of Agriculture and Rural Affairs, Institute of Feed Research, Chinese Academy of Agricultural
Sciences, Beijing 100081, China; caasgs20141114@126.com (S.G.); qiukai@caas.cn (K.Q.);
zhanghaijun@caas.cn (H.Z.); wangjing@caas.cn (J.W.)
2 Beijing Agricultural Products Quality and Safety Center, the Ministry of Agriculture and Rural Affairs,
Beijing 100020, China; 13521296211@163.com
3 Animal Science and Technology College, Beijing University of Agriculture, Beijing 102206, China
* Correspondence: buaqxl@126.com (X.Q.); wushugeng@caas.cn (S.W.);
Tel.: +86-10-80799133 (X.Q.); +86-10-82106097 (S.W.)

Simple Summary: Improving the production performance and egg quality of laying hens in the late-
laying period has become an urgent problem. This study confirmed that dietary supplementation of
25-hydroxycholecalciferol (25-OHD3 ) instead of vitamin D3 (VD3 ) improved production performance
and egg quality, maintained the jejunal morphology, and enhanced tibia quality by increasing the
concentration of circulating 25-OHD3 and Ca absorption in late-laying-period hens. The superiority
of 25-OHD3 over VD3 indicates that it has the potential to prevent an age-induced decrease in
production performance and osteoporosis in laying hens, thereby contributing to extending the
feeding cycle of laying hens.
Citation: Gao, S.; Qiu, K.; Zheng, J.;
Zhang, H.; Wang, J.; Qi, X.; Wu, S. Abstract: This study aimed to investigate whether a dietary 25-OHD3 addition improved the per-
Dietary 25-Hydroxycholecalciferol formance, egg quality, blood indexes, antioxidant status, jejunal morphology, and tibia quality of
Supplementation as a Vitamin D3 aged laying hens compared to a dietary VD3 addition. A total of 270 Hy-Line Brown laying hens
Substitute Improves Performance, at 55 wk of age were randomly assigned to three dietary treatments with six replicates (15 birds
Egg Quality, Blood Indexes, Jejunal per replicate with 3 birds per cage). Chickens were fed a corn–soybean meal diet supplementa-
Morphology, and Tibia Quality in
tion of 4000 IU/kg VD3 (control group), 50 µg/kg 25-OHD3 and 2000 IU/kg VD3 (experimental
Late-Phase Laying Hens. Animals
group 1), or 50 µg/kg 25-OHD3 and 4000 IU/kg VD3 (experimental group 2) for 12 weeks. The
2024, 14, 878. https://doi.org/
results demonstrated that 25-OHD3 caused a significant increase in the laying rate, especially in the
10.3390/ani14060878
50 µg/kg 25-OHD3 + 2000 IU/kg VD3 group; the laying rate reached the maximum compared with
Academic Editor: Fulvia Bovera other groups after 12 weeks (p < 0.05). However, there were no significant effects on the average
Received: 8 February 2024
egg weight, average daily feed intake, or feed-to-egg ratio (p > 0.05). A dietary supplementation of
Revised: 5 March 2024 50 µg/kg 25-OHD3 and 2000 IU/kg VD3 provided an improved eggshell strength, thick albumen
Accepted: 5 March 2024 height, and Haugh unit after 12 weeks (p < 0.05). Further analysis of the blood indexes showed
Published: 13 March 2024 that alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, calcium, and phos-
phorus were enhanced significantly in the 50 µg/kg 25-OHD3 + 2000 IU/kg VD3 group, while the
content of total bilirubin decreased significantly (p < 0.05). In addition, the 25-OHD3 addition in diets
improved the calcium and phosphorus contents in the serum (p < 0.05). The concentrations of 25-
Copyright: © 2024 by the authors.
OHD3 , parathyroid hormones, follicle-stimulating hormone, and progesterone were increased in the
Licensee MDPI, Basel, Switzerland.
50 µg/kg 25-OHD3 + 2000 IU/kg VD3 group, and the levels of cortisol, calcitonin, bone gla protein,
This article is an open access article
distributed under the terms and
and endotoxin in the serum reached a minimum in the 50 µg/kg 25-OHD3 + 4000 IU/kg VD3 group
conditions of the Creative Commons (p < 0.05), which constitutes an advantage for the aged laying hens. The antioxidant enzyme activities
Attribution (CC BY) license (https:// and free radical scavenging abilities in the 50 µg/kg 25-OHD3 + 2000 IU/kg VD3 group increased
creativecommons.org/licenses/by/ markedly, and the MDA level decreased significantly in the 50 µg/kg 25-OHD3 + 4000 IU/kg VD3
4.0/). group (p < 0.05). Improvements in jejunal morphology and intestinal integrity resulted in an increased

Animals 2024, 14, 878. https://doi.org/10.3390/ani14060878 https://www.mdpi.com/journal/animals

Animals 2024, 14, 878 2 of 15

villi-length-to-crypt-depth ratio in the 50 µg/kg 25-OHD3 + 2000 IU/kg VD3 group (p < 0.05). Dietary
50 µg/kg 25-OHD3 and 2000 IU/kg VD3 additions improved the tibia quality, including fresh tibia
weight, strength, mineral content (Ca), and trabeculae area (p < 0.05). Taken together, compared with
the dietary VD3 addition, dietary supplementation of 25-OHD3 supported a stable physiological
status for sustained egg production, egg quality, and bone quality in late-phase laying hens, and the
addition levels of 50 µg/kg 25-OHD3 and 2000 IU/kg VD3 had the best effect. Therefore, this could
provide a theoretical basis for the use of 25-OHD3 as a substitute forVD3 .

Keywords: 25-hydroxycholecalciferol; calcium absorption; performance; tibia health; late-phase

laying hens

1. Introduction
The laying period from 45 weeks of age to 72 weeks of age is often referred to as the
late phase of hen laying. This cycle of production is often linked with increased reactive
oxygen species (ROS) production, declined production performance, reduced antioxidant
capacity, low secretion of reproductive hormones, and impaired intestinal oxidation status,
which culminate in poor eggshell quality/irregular shell formation [1,2]. Laying hens
absorb calcium from the intestine or via bone resorption. About 30–40% of the calcium
released from bones is deposited into the eggshells of birds in the late-laying phase, which
is reflected in a lower bone mineral content and density [3,4]. Laying hens in the peak
laying period, reared on a low-Ca (1.5%) diet, had higher rates of broken eggs and lower
body weights, feed consumption, and laying rates compared to birds fed normal-Ca (3.9%)
diets [5]. In the late-production phase, increasing dietary Ca from 3.5% to 4.5% linearly
improved the laying rate of birds [6]. Mineral reserves are critical for eggshell quality
and bone integrity in older laying hens since intestinal Ca absorption diminishes with
age [7]. Consequently, Ca supplementation is required throughout the production cycle of
laying hens.
Moreover, the skeletal health of laying hens has gained research focus. According to a
previous study, 30% of laying hens suffer bone fractures before slaughter, reducing their
economic value [8]. Currently, the cage housing system is the most popular raising strategy
for layers in China due to its cost advantages, i.e., because there are no significant effects
on egg deposition patterns and egg quality, except for improving the state of the feathers
of laying hens compared with some welfare cages [9]. However, the cage leg problem
may be a challenge for old laying hens because the late phase of production is linked with
incidences of osteoporosis [10]. Also, caged hens with high laying rates develop “cage layer
fatigue” (CLF), which often leads to structural bone loss and fractures in various bone areas
at the end of the laying cycle [11]. Higher laying rates and low-Ca diets may cause loss of
parenchymal bone and osteoporosis, the main bone disease of laying hens [11,12].
Optimal performance in aged laying hens requires nutritional interventions that
can maintain antioxidant status, Ca requirements, and homeostasis, and reduce negative
impacts on performance, egg quality, and bone health. Vitamin D3 (VD3 ), also known as
cholecalciferol, promotes the intestinal absorption of Ca and regulates mineral and skeletal
homeostasis [13,14]. Previously, supplementing laying hens with VD3 (from 1681 IU/kg to
35,014 IU/kg) from 0 to 68 weeks increased the laying rate, egg quality attributes (Haugh
unit, eggshell breaking strength), and bone quality linearly, but these positive effects were
reversed at 68,348 IU/kg [15]. The inclusion of an appropriate amount of VD3 in the diet
can boost the laying rate and tibial breaking strength [16]. These findings suggest that
dietary VD3 supplementation increased the laying performance within a certain inclusion
range. However, dietary supplementation of higher levels VD3 may have a detrimental
effect on the performance of laying hens.
Animals 2024, 14, 878 3 of 15

VD3 is activated and converted into an active metabolite (25-OHD3 ) in the liver [17],
but the conversion may be impaired in old laying hens. 25-OHD3 had higher biological
activity than VD3 , less side effects, and greater stability than 1,25-dihydroxycholecalciferol,
and dietary 25-OHD is characterized by a better strong affinity and absorption efficiency
with Vitamin D binding protein [18]. Nowadays, 25-OHD3 has been validated as a source of
VD3 and is widely utilized in poultry production. Previous studies have shown that the ad-
dition of 25-OHD3 in the diet has positive effects on the production performance and bone
health of laying hens. A study showed that long-term (from 0–95 weeks) replacement of
VD3 with 25-OHD3 (equivalent from VD3 ) significantly increased the circulating 25-OHD3
concentration, improved bone health, and provided more space for mineral deposition
during the late-production phase of laying hens [19]. However, another study showed
that compared with 62.5 µg/kg doses of VD3 , supplementary 125 µg/kg doses of VD3
or 25-OHD3 improved the performance and egg quality, while there were no remarkable
differences among the two groups [20].
Although 25-OHD3 is a bioactive dietary form (1 µg of cholecalciferol is equivalent to
40 IU of VD3 ), the effects of dietary 25-OHD3 supplementation as a vitamin D3 substitute
on late-laying hens are unknown. Given the aforementioned findings, this study aimed
to verify the effects of dietary 25-OHD3 as a VD3 alternative on performance, egg quality,
blood indices, jejunal morphology, and tibia quality. To further explore whether dietary
supplementation of 25-OHD3 can be used as a nutritional strategy to improve bone health
in aging progress by promoting the absorption and deposition of calcium and phosphorus.

2. Materials and Methods

The experimental protocol used in this study was approved by the Animal Care and
Use Committee (IFR-CAAS-20220401) of the Institute of Feed Research of the Chinese
Academy of Agriculture Science.

2.1. Birds and Housing

During the feeding trial, laying hens were randomly housed in 3-tier battery cages
(40 cm × 40 cm × 35 cm) and exposed to 16 h of incandescent light and 8 h of darkness on
a daily basis, and the light intensity was 20 lux. The birds were fed twice daily (8:00 and
14:00) and allowed ad libitum access to water and treatment diets during the experimental
period. The average temperature in the laying hen house was 21 ± 3 ◦ C with natural
ventilation and the room’s relative humidity at 50–80%. The birds were in good health
throughout the feeding trial.

2.2. Experimental Design and Diets

A total of 270 Hy-Line Brown laying hens (55 weeks old, 2.08 ± 0.09 kg body weight)
were randomly assigned to three treatment groups with six replicates per treatment (15 lay-
ing hens per replicate). The control group was fed a basal diet supplemented with
4000 IU/kg VD3 . Experimental group 1 was fed a basal diet with a supplementation
of 50 µg/kg 25-OHD3 and 2000 IU/kg VD3 . Experimental group 2 was fed a basal diet
supplemented with 50 µg/kg 25-OHD3 and 4000 IU/kg VD3 . The product with 0.05% of
25-OHD3 was purchased from Zhejiang Jin Langbo Pharmaceutical Co., Ltd. (Xinchang,
China) and prepared by using the biotransformation method. The composition and nutrient
levels of the corn–soybean meal-based diets are shown in Table 1. All hens were fed a basal
diet for 1 week and then assigned to dietary treatments for 12 weeks.
Animals 2024, 14, 878 4 of 15

Table 1. Composition and nutrient level of basal diet.

Ingredients Content, % Nutrient Level 2

Corn 63.65 CP, % 16.50
Soybean meal 25.41 Metabolizable energy 2 , MJ/kg 11.30
Soybean oil 0.11 Calcium, % 3.50
Stone powder 9.18 Total phosphorus, % 0.49
Calcium hydrophosphate 0.90 Available phosphorus, % 0.29
Salt 0.30 Met + Cys, % 0.65
DL-Methionine 0.18 Lysine, % 0.79
Premix 1 0.27 Threonine, % 0.56
Total 100.00 Tryptophan, % 0.18
1 The premix provided the following per kilogram of the diet: VA 12,500 IU, VE 15 IU, VK 2 mg, VB1 1 mg, VB2
8.5 mg, VB6 8 mg, VB12 5 mg, calcium pantothenate 50 mg, nicotinic acid 32.5 mg, biotin 2 mg, folic acid 5 mg,
choline 500 mg, Mn 65 mg, I 1 mg, Fe 60 mg, Cu 8 mg, Zn 66 mg, and phytase 350 IU. 2 Nutrition levels were all
calculated values. Metabolizable energy (ME) was calculated according to experimental diet composition and ME
values of feedstuffs on poultry in the Chinese Feed Database (2018 Twenty-Ninth Edition).

2.3. Sample Collection and Measurements

At weeks 4, 8, and 12 of the trial, five eggs were randomly selected from each replicate;
thus, a total of 270 eggs were used for the egg quality determination during the three
assessment periods, and egg quality detection was completed within 3 h after egg collection
(the storage temperature was 24–26 ◦ C). At the end of the experiment period, 6 birds per
treatment (1 bird per replicate) were randomly selected and fasted for 8 h prior to slaughter.
Blood samples were collected from the wing vein of each bird, and the serum was separated
by centrifugation at 3000× g for 10 min and stored at −20 ◦ C for biochemical parameter
analysis. The selected birds were sacrificed by cervical dislocation, and the jejunum samples
were collected and fixed in a 4% paraformaldehyde solution for 48 h. Also, the left and right
tibias of each bird were collected and cleaned of all tissue (muscle, tendon, and fat). The
left tibia was fixed with 4% paraformaldehyde and decalcified with 10% EDTA solution.
The right fresh tibia samples were weighed with an electronic scale (accuracy of 0.01 g)
after removing the fat and tissue.

2.4. Production Performance and Egg Quality Analysis

Production performances (egg number, total egg weight, feed intake, and unqualified
eggs (egg weight < 50 g or 75 g)) were recorded daily for each replicate, accordingly. The
feed-to-egg ratio was calculated as the ratio of feed intake (grams) to the total egg weight
(grams). The laying rate was calculated as the total number of eggs/total number of hens
× 100%. The average egg weight, average daily feed intake, laying rate, and feed-to-egg
ratio were calculated for wks 1–4, wks 5–8, and wks 9–12. The average egg weight was
calculated as the total egg weight/total number of eggs. The egg weight, egg yolk weight,
and albumen weight were measured by an electronic scale (accuracy 0.01). The ratio of
egg yolk or albumen was calculated as their weight/egg weight × 100%. The length
and width of eggs were measured using an electronic vernier caliper (Shanghai Shenhan
Measuring Tools Co., Ltd., Shanghai, China), and the egg shape index was calculated using
the length/width ratio. Also, the eggshell strength was determined with an eggfore reader
(ORKA Food Technology Ltd., Ramat HaSharon, Israel). In addition, the Haugh unit and
thick albumen height were determined with an automated egg quality analyzer (ORKA
Food Technology Ltd., Ramat HaSharon, Israel).

2.5. Blood and Serum Biochemical Parameters Analyses

The blood biochemical indices include the activities of alanine aminotransferase (ALT),
aspartate aminotransferase (AST), and alkaline phosphatase (ALP). Moreover, the levels
of albumin (ALB), uric acid (UA), creatinine (CRE), glucose (GLU), total bilirubin (T-BIL),
calcium (Ca), and phosphorus (P) were determined by an ultraviolet spectrophotometer.
These assays were performed using a commercial kit (Shanghai Kehua Bio-Engineering
Animals 2024, 14, 878 5 of 15

Co., Ltd., Shanghai, China), and all protocols were followed in accordance with the manu-
facturer’s instructions.
The activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), cata-
lase (CAT), the abilities of scavenging radicals, the total antioxidant capacity (T-AOC), and
the product of lipid metabolism (MDA) concentration were analyzed using the commer-
cial kits (Nanjing Jiancheng Bio-Engineering Institute, Nanjing, China) by a microplate
reader. In addition, the hormone levels included 25-OHD3 , follicle-stimulating hormone
(FSH), testosterone (T), progesterone, parathyroid hormone (PTH), bone gla protein (BGP),
calcitonin (CT), carbonic anhydrase (CA), cortisol (COR), diamine oxidase (DAO), and en-
dotoxin (ET). These assays were performed using the chicken ELISA kits (Nanjing Jiancheng
Bio-Engineering Institute, Nanjing, China) according to the instructions.

2.6. Intestinal Morphology Analysis

Intestinal morphology analysis was conducted using a previously described method [21].
Briefly, jejunum tissues were dehydrated, embedded in paraffin, and sectioned into 4 µm
thick sections for histological examination. After HE (hematoxylin–eosin) staining, each
stained section was photographed under a 40× magnification with a digital microscope
(Olympus Corporation, Tokyo, Japan), and three typical fields of view were selected for
each stained section. The villus height and crypt depth were measured using the medical
image analysis software ImagePro-Plus 7.0 (Media Cybernetics lnc., Rockville, MD, USA).
Finally, the ratio of villi-length-to-crypt-depth (V/C) was calculated.

2.7. Tibial Quality Analysis

The left tibia samples were fixed with 4% paraformaldehyde and decalcified with
10% EDTA solution, then dehydrated and embedded in paraffin. The paraffin blocks
were sectioned into 5 µm thick sections, stained with toluidine blue, and a histological
observation was performed under the scan at 200× magnification. The number of bone
trabeculae and area of bone trabeculae were evaluated by the medical image analysis
software ImagePro-Plus 7.0 (Media Cybernetics lnc., Rockville, MD, USA).
Furthermore, the tibia strength was measured with a texture analyzer (Food Tech-
nology Corporation, Sterling, VA, USA). A three-point bending test of the metaphyseal
tibia was done with a supporting distance of 30 mm and a test speed of 10 mm/min [22].
According to previous study methods, the ash, calcium (Ca), and phosphorus (P) contents
of tibia were determined [23]. Briefly, the bone samples were placed in an oven at 105 ◦ C for
48 h to eliminate moisture and then weighed. Secondly, after defatting with petroleum ether
(Sinopharm Chemical Reagent Co., Ltd., Beijing, China), the dried bone was carbonized
at 600 ◦ C for 3 h. Next, after ashing at 550 ◦ C for 12 h in a muffle furnace, samples were
ground into powder, and the percentage of ash was determined.
For further analysis, a tibial powder sample (0.2 g) was added to the conical flask,
5 mL of nitric acid (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was fully
mixed with the sample, and it was digested at 220 ◦ C in an electric sand bath for 3 h until
there was no turbidity in the solution. After cooling at room temperature, the samples were
diluted 625 times with ultrapure water and filtered through a 0.22 µm filter membrane.
The contents of Ca and P in the samples were detected by an inductively coupled plasma
atomic emission spectrometer (Agilent Technologies lnc., Palo Alto, CA, USA).

2.8. Statistical Analysis

Statistical analysis was performed using SPSS 20.0 (IBM Corp., Armonk, NY, USA).
The data relating to the effect of dietary 25-OHD3 levels on performance, egg quality, blood
indexes, intestinal morphology, and tibia quality in the late-laying period were analyzed by
one-way ANOVA, the means were compared using Tukey’s multiple test, and significant
differences were considered at p < 0.05.
Animals 2024, 14, 878 6 of 15

3. Results
3.1. Production Performance
Production performance is one of the important indicators for evaluating poultry
production efficiency. The effects of dietary supplementation of 25-OHD3 on the produc-
tion performance of laying hens are listed in Table 2. The results showed that dietary
supplementation of 25-OHD3 significantly increased the laying rate (p < 0.05) compared to
the control group. Particularly, the dietary addition of 50 µg/kg 25-OHD3 and 2000 IU/kg
VD3 significantly increased the laying rate at weeks 8 and 12 compared to other groups
(p < 0.05). However, there were no significant effects of the dietary 25-OHD3 addition on
the average egg weight, average daily feed intake, and feed-to-egg ratio related to the
control group throughout the feeding trial (p > 0.05). In addition, no mortality occurred
throughout the test period.

Table 2. Effects of dietary supplementation of 25-OHD3 on performance of laying hens.

25-OHD3 Levels, µg/kg

Items Time/Week 0 50 SEM p-Value
4000 IU VD3 2000 IU VD3 4000 IU VD3
1–4 62.04 61.99 61.88 0.20 0.95
Average egg weight, g 5–8 61.32 61.47 61.16 0.27 0.91
9–12 61.14 61.91 62.12 0.55 0.60
1–4 123.68 121.40 122.23 0.70 0.43
Average daily feed
5–8 121.24 124.34 119.44 0.98 0.11
intake, g
9–12 125.35 121.78 119.78 2.22 0.61
1–4 2.09 2.13 2.07 0.02 0.54
Feed-to-egg ratio 5–8 2.12 2.24 2.07 0.03 0.08
9–12 2.21 2.06 2.17 0.03 0.11
1–4 87.88 87.58 90.79 0.62 0.05
Laying rate, % 5–8 86.63 b 89.54 a 87.88 ab 0.19 0.01
9–12 78.19 b 84.76 a 80.47 b 0.83 <0.01
In the same row, values with different small letter superscripts mean significant difference (p < 0.05).

3.2. Egg Quality Assessment

In this study, the effects of dietary 25-OHD3 supplementation on the egg quality of
laying hens are listed in Table 3. The enhancement effects of the dietary 50 µg/kg 25-OHD3
and 4000 IU/kg VD3 addition on eggshell strength was obvious at the end of week 8, and
eggshell strength improved significantly in the 50 µg/kg 25-OHD3 + 2000 IU/kg VD3
group at week 12 (p < 0.05). We further found that dietary supplementation of 25-OHD3
caused a significant increase in the thick albumen height and Haugh unit (p < 0.05), and the
value reached the maximum in the 50 µg/kg 25-OHD3 + 2000 IU/kg VD3 groups at week
12. Notably, there were no significant effects on the eggshell strength, thick albumen height,
and Haugh unit in the 50 µg/kg 25-OHD3 + 2000 IU/kg VD3 group or 50 µg/kg 25-OHD3
+ 4000 IU/kg VD3 group. Moreover, there were no significant effects of dietary treatments
on the egg yolk weight, albumen weight, egg yolk rate, albumen rate, and egg shape index
(p > 0.05).

Table 3. Effects of dietary supplementation of 25-OHD3 on egg quality of laying hens.

25-OHD3 Levels, µg/kg

Items Time/Week 0 50 SEM p-Value
4000 IU VD3 2000 IU VD3 4000 IU VD3
4 64.43 64.39 62.87 0.55 0.54
Egg weight, g 8 61.03 61.56 61.18 0.32 0.80
12 62.09 62.64 62.03 0.54 0.89
Animals 2024, 14, 878 7 of 15

Table 3. Cont.

25-OHD3 Levels, µg/kg

Items Time/Week 0 50 SEM p-Value
4000 IU VD3 2000 IU VD3 4000 IU VD3
4 17.02 17.04 16.97 0.12 0.97
Egg yolk weight, g 8 16.40 16.48 16.26 0.18 0.90
12 16.24 15.88 16.12 0.13 0.53
4 26.41 26.50 26.47 0.30 0.99
Egg yolk ratio,% 8 26.86 26.76 26.58 0.28 0.93
12 25.86 25.77 25.95 0.21 0.94
4 41.11 41.13 40.11 0.49 0.65
Albumen weight, g 8 38.96 39.39 39.11 0.28 0.83
12 40.39 40.48 39.93 0.42 0.86
4 63.82 63.84 63.77 0.31 1.00
Albumen ratio,% 8 63.84 63.99 63.92 0.33 0.98
12 65.04 64.63 64.35 0.23 0.51
4 1.23 1.24 1.24 0.005 0.31
Egg shape index 8 1.31 1.32 1.32 0.005 0.99
12 1.30 1.29 1.28 0.005 0.46
4 37.60 39.32 40.00 0.45 0.07
Eggshell strength, N 8 33.32 b 33.19 b 36.66 a 0.44 <0.01
12 33.36 b 35.14 a 34.21 ab 0.28 0.02
4 6.44 b 6.99 a 7.09 a 0.09 <0.01
Thick albumen height,
8 6.16 b 7.07 a 7.07 a 0.12 <0.01
mm
12 6.42 b 7.50 a 7.45 a 0.14 <0.01
4 75.89 b 81.33 a 82.37 a 0.73 <0.01
Haugh unit 8 77.02 b 83.53 a 83.30 a 0.85 <0.01
12 79.17 b 85.32 a 84.77 a 0.74 <0.01
In the same row, values with different small letter superscripts mean significant difference (p < 0.05).

3.3. Serum Biochemistry Analysis

The effects of dietary 25-OHD3 supplementation on the serum biochemical indices
are presented in Table 4. There were notable effects of the dietary addition of 50 µg/kg
25-OHD3 and 2000 IU/kg VD3 on the ALT activity, ALB level, and P level (p < 0.05). More-
over, Ca levels in the 50 µg/kg 25-OHD3 + 4000 IU/kg VD3 groups enhanced significantly
(p < 0.05). However, the T-BIL level in the 50 µg/kg 25-OHD3 + 2000 IU/kg VD3 group
and the ALP level in the 50 µg/kg 25-OHD3 + 4000 IU/kg VD3 group declined remarkably,
respectively (p < 0.05). Moreover, no remarkable differences in the activity of AST were
observed among the dietary groups (p > 0.05). Moreover, compared to the control group,
the levels of UA, CRE, and GLU in serum remained similar across the dietary treatments
(p > 0.05).

Table 4. Effects of dietary supplementation of 25-OHD3 on serum biochemical indicators of laying hens.

25-OHD3 Levels, µg/kg

Items 0 50 SEM p-Value
4000 IU VD3 2000 IU VD3 4000 IU VD3
b a
ALT, U/L 36.32 41.51 37.17 b 0.77 <0.01
AST, U/L 74.12 75.67 73.64 0.76 0.56
ALP, U/L 331.83 a 298.09 b 291.82 b 5.42 <0.01
ALB, g/L 24.32 b 30.85 a 29.51 a 0.88 <0.01
UA, umol/L 252.31 241.99 252.09 3.73 0.46
CRE, umol/L 73.89 77.16 74.43 0.78 0.20
Animals 2024, 14, 878 8 of 15

Table 4. Cont.

25-OHD3 Levels, µg/kg

Items 0 50 SEM p-Value
4000 IU VD3 2000 IU VD3 4000 IU VD3
GLU, mmol/L 12.52 12.33 13.10 0.24 0.42
T-BIL, umol/L 32.69 a 26.94 b 30.07 ab 0.76 <0.01
Ca, mmolL 2.45 b 2.64 ab 2.75 a 0.05 0.02
P, mmol/L 2.40 b 2.84 a 2.73 a 0.05 <0.01
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; ALB,
albumin; UA, uric acid; CRE, creatinine; GLU, glucose; T-BIL, total bilirubin; Ca, calcium; P, phosphorus. In the
same row, values with different small letter superscripts mean significant difference (p < 0.05).

3.4. Serum Hormone Concentration

The effects of dietary 25-OHD3 supplementation on the concentration of serum hor-
mones are shown in Table 5. The concentration of 25-OHD3 in the serum was significantly
increased in the 50 µg/kg 25-OHD3 + 2000 IU/kg VD3 group (p < 0.05). In addition, a
similar trend for enhancement effects in the 50 µg/kg 25-OHD3 + 2000 IU/kg VD3 bird-fed
group was notable for concentrations of hormones, including FSH, progesterone, T, and
PTH (p < 0.05). Nevertheless, it is noteworthy to mention that the dietary inclusion of 25-
OHD3 remarkably decreased concentrations of -COR, BGP, CT, and ET (p < 0.05) compared
to the control group. There were no significant effects in the concentrations of CA and DAO
among all the groups (p > 0.05).

Table 5. Effects of dietary supplementation of 25-OHD3 on hormone levels in serum of laying hens.

25-OHD3 Levels, µg/kg

Items 0 50 SEM p-Value
4000 IU VD3 2000 IU VD3 4000 IU VD3
b a
25-OHD3 , ng/mL 5.34 5.81 5.55 ab 0.08 0.02
FSH, IU/L 10.09 c 16.31 a 13.16 b 0.65 <0.01
T, nmol/L 1.14 b 1.39 a 1.47 a 0.04 <0.01
Progesterone, nmol/L 8.01 b 9.04 a 8.07 b 0.15 <0.01
CA, ng/mL 5.21 5.60 5.40 0.07 0.06
COR, ng/mL 26.81 a 18.25 b 10.20 c 1.65 <0.01
DAO, ng/mL 7.79 7.51 7.26 0.10 0.10
ET, ng/mL 11.30 a 11.37 a 9.75 b 0.22 <0.01
PTH, pg/mL 16.88 ab 17.26 a 15.09 b 0.38 <0.01
BGP, ng/mL 1.50 a 1.29 b 1.20 b 0.04 <0.01
CT, pg/mL 4.28 a 4.04 b 3.95 b 0.04 <0.01
Abbreviations: FSH, follicle-stimulating hormone; T, testosterone; CA, carbonic anhydrase; COR, cortisol; DAO,
diamine oxidase; ET, endotoxin; PTH, parathyroid hormone; BGP, bone gla protein; CT, calcitonin. In the same
row, values with different small letter superscripts mean significant difference (p < 0.05).

3.5. Antioxidant Capacity

The effects of dietary 25-OHD3 supplementation on the serum antioxidant parameters
are presented in Table 6. The activities of GSH-Px, CAT, and SOD in the 50 µg/kg 25-OHD3
+ 2000 IU/kg VD3 groups were higher than that in the control group, while the MDA level
was drastically reduced in the 50 µg/kg 25-OHD3 + 4000 IU/kg VD3 group (p < 0.05).
Moreover, the abilities of scavenging superoxide anions and scavenging hydroxyl radicals
were enhanced markedly (p < 0.05) in the treatment groups compared to the control group.
However, there were no significant effects on the T-AOC levels among the groups (p > 0.05).
Thus, dietary supplementation of 50 µg/kg 25-OHD3 and 2000 IU/kg VD3 provided the
highest antioxidant capacity.
 to the control group. However, there were no significant effects on the T-AOC levels
among the groups (p > 0.05). Thus, dietary supplementation of 50 µg/kg 25-OHD3 and
2000 IU/kg VD3 provided the highest antioxidant capacity.
Animals 2024, 14, 878 Table 6. Effects of dietary supplementation of 25-OHD3 on antioxidant abilities in serum of9laying
of 15
hens.

25-OHD3 Levels,
Table 6. Effects of dietary supplementation μg/kg on antioxidant abilities in serum of laying hens.
of 25-OHD 3
Items 0 50 SEM p-Value
25-OHD
4000 IU VD3 3 Levels,
2000µg/kg
IU VD3 4000 IU VD3
GSH-Px,
Items U/mL 0 519.48 c 893.23
50 a 814.94 b SEM 39.37 <0.01
p-Value
CAT, U/L 4000 IU VD3 5.46 2000 IU VD3 19.95 4000 IU VD3 11.86
c a b 1.45 <0.01
SOD, U/mL 14.92 b 17.81 a 15.21 b 0.41 0.01
GSH-Px, U/mL 519.48 c 893.23 a 814.94 b 39.37 <0.01
MDA,
CAT, U/L nmol/mL 5.46 c 21.62 a
19.95 a 8.24 b
11.86 b 7.90 b
1.45 1.57 <0.01
<0.01
Hydroxyl radical scavenging14.92 b
SOD, U/mL 17.81 a
15.21 b 0.41 0.01
238.15 b 248.32 a 255.02 a 2.05 <0.01
ability, U/mL
MDA, nmol/mL 21.62 a 8.24 b 7.90 b 1.57 <0.01
Hydroxyl radical
Superoxide anion scavenging238.15 b a a
scavenging ability, U/mL 435.51 b 248.32 527.77 a 255.02 516.36 a 2.05 10.42 <0.01
<0.01
ability, U/mL
Superoxide anion
435.51 b 527.77 a 516.36 a 10.42 0.42 <0.01
scavengingT-AOC, U/mL
ability, U/mL 18.99 20.49 21.57 0.07
T-AOC, U/mL Abbreviations:
18.99 GSH-Px, glutathione
20.49 peroxidase; CAT, catalase; SOD,0.42
21.57 superoxide dismutase;
0.07 MDA,
malondialdehyde; T-AOC, total antioxidant capacity. In the same row, values with different
Abbreviations: GSH-Px, glutathione peroxidase; CAT, catalase; SOD, superoxide dismutase; MDA, malondialde-
small
letter superscripts mean significant difference (p < 0.05).
hyde; T-AOC, total antioxidant capacity. In the same row, values with different small letter superscripts mean
significant difference (p < 0.05).
3.6. Intestinal Morphology
3.6. Intestinal Morphologyof the jejunum in laying hens is shown in Figure 1. The dietary
The morphology
The morphology
addition of the jejunum
of 25-OHD3 presented in laying
an intact hensstructure
intestinal is shown in Figure
with longer 1.
villiThe
anddietary
regular
addition of 25-OHD presented an intact intestinal structure with longer villi
crypt morphology 3(p < 0.05) compared to the control group. Additionally, the under 50 and regular
crypt
µg/kg morphology
25-OHD3 + 2000 (p < IU/kg
0.05) compared to the control
VD3 bird groups, group. toAdditionally,
in comparison the under
the control group, had a
50significant
µg/kg 25-OHDreduction
3 + 2000 IU/kg VD bird groups, in comparison to the control group,
in crypt depth3 and notable increases in the villi-length-to-crypt-
had a significant
depth ratio (p < reduction
0.05). in crypt depth and notable increases in the villi-length-to-crypt-
depth ratio (p < 0.05).

Figure 1. Effects of dietary supplementation of 25-OHD3 on intestinal morphology of laying hens.

Figure 1. Effects of dietary supplementation of 25-OHD3 on intestinal morphology of laying hens.
(A) The jejunum was stained with HE solution (300 microns, ×40). (B) Crypt depth. (C) Villus
(A) The jejunum was stained with HE solution (300 microns, ×40). (B) Crypt depth. (C) Villus height.
height. (D) Villi-length-to-crypt-depth ratio. The different superscript small letters were judged as a
significant difference (p < 0.05).

3.7. Tibia Quality

As shown in Figure 2, the Ca content in the 50 µg/kg 25-OHD3 + 2000 IU/kg VD3
group or 50 µg/kg 25-OHD3 + 4000 IU/kg VD3 groups were remarkably increased (p < 0.05)
compared to the control group, but there were no significant variations for the content of
 significant difference (p < 0.05).

3.7. Tibia Quality

As shown in Figure 2, the Ca content in the 50 µg/kg 25-OHD3 + 2000 IU/kg VD3 group
Animals 2024, 14, 878 or 50 µg/kg 25-OHD3 + 4000 IU/kg VD3 groups were remarkably increased (p <100.05) of 15
compared to the control group, but there were no significant variations for the content of
ash and P (p > 0.05) in the tibia samples. Moreover, a histopathology evaluation of the tibia
revealed
ash and Pthe(p presence
> 0.05) inofthe
a cortical bone structure
tibia samples. Moreover, and trabeculae in the
a histopathology tibia as closely
evaluation of the
arranged. The number and area of trabeculae in the 50 µg/kg 25-OHD 3 + 2000 IU/kg VD3
tibia revealed the presence of a cortical bone structure and trabeculae in the tibia as closely
group wereThe
arranged. significantly
number and increased
area of relative to the
trabeculae control
in the group25-OHD
50 µg/kg (p < 0.05).+ In addition,
2000 IU/kg
3
dietary 50 µg/kg 25-OHD 3 and 2000 IU/kg VD3 supplementation increased the fresh tibia
VD3 group were significantly increased relative to the control group (p < 0.05). In addition,
weight
dietary and strength
50 µg/kg (p < 0.05).
25-OHD 3 and 2000 IU/kg VD3 supplementation increased the fresh tibia
weight and strength (p < 0.05).

Figure 2. Effects of dietary supplementation of 25-OHD3 on tibia quality of laying hens. The content
Figure 2. Effects of dietary supplementation of 25-OHD3 on tibia quality of laying hens. The content
of (A) ash, (B) Ca, and (C) P in the tibia. (D) Effect of dietary 25-OHD on tibia histopathology of
of (A) ash, (B) Ca, and (C) P in the tibia. (D) Effect of dietary 25-OHD33 on tibia histopathology of
laying hens:
laying hens: the
the medullary
medullary trabecular
trabecular bone
bone was
was stained
stainedwith
withtoluidine
toluidineblue solution(×
bluesolution 200). (E)
(×200). Fresh
(E)Fresh
tibia weight. (F) Tibia strength. (G) The number of trabecular. (H) The area of trabeculae bone.
tibia weight. (F) Tibia strength. (G) The number of trabecular. (H) The area of trabeculae bone. The The
different superscript
different superscript small
small letters
letters were
were judged
judged as
as aa significant
significant difference
difference (p
(p < 0.05).
< 0.05).

4. Discussion
4. Discussion
Achieving optimal laying rates, eggshell quality, and bone health are major issues
Achieving optimal laying rates, eggshell quality, and bone health are major issues of
of concern for the poultry industry with respect to laying hens in the late phase of the
concern for the poultry industry with respect to laying hens in the late phase of the
production cycle. Previous reports showed that the laying rate and egg quality (including
production cycle. Previous reports showed that the laying rate and egg quality (including
the eggshell strength and thickness) decline with the age increase of hens [24]. Consequently,
the eggshell strength and thickness) decline with the age increase of hens [24].
about 10–15% of produced eggs are lost due to poor eggshell quality [25]. Laying hens
Consequently, about 10–15% of produced eggs are lost due to poor eggshell quality [25].
with high egg production rates may develop avian osteoporosis due to the resorption
Laying
of both hens with high
medullary and egg production
structural bone rates may develop
for eggshell avian osteoporosis
development during the due
late to the
phase
resorption of both medullary and structural bone for eggshell development during
of the cycle [11]. Interestingly, nutritional strategies can mitigate the adverse impacts the
late phase
of age of the cycleperformance,
on production [11]. Interestingly, nutritional
bone health, andstrategies canduring
egg quality mitigate
thethe adverse
late-laying
phase. The current study provides support for the dietary addition of 25-OHD3 as a VD3
substitute to enhance performance, egg quality, physiology, and bone health in birds at the
late-laying phase.
In the current study, dietary supplementation of 25-OHD3 significantly increased
the laying rate compared to the VD3 -supplemented group. The previous study reported
that dietary supplementation of an equal amount of 25-OHD3 had similar effects to VD3 ’s
significantly increased laying rate and decreased unqualified egg rate [20]. In another study,
dietary supplementation of 25-OHD3 increased the production rate and feed conversion
Animals 2024, 14, 878 11 of 15

ratio compared with a dietary VD3 addition in aged laying hens [13]. Thus, the dietary
addition of 25-OHD3 or 25-OHD3 and VD3 combination exerted a superior positive effect
on the laying rate of late-laying phase hens compared to the normal VD3 group, which is
due to more demand for more active 25-OHD3 to maintain the production performance in
this laying phase. In another study, dietary 25-OHD3 did not influence the laying rate, egg
weight, and feed efficiency of laying hens [26,27]. Long-term dietary supplementation of
25-OHD3 at 69 or 125 µg/kg as a substitute to VD3 had no significant effects on the laying
rate, egg weight, and feed efficiency but enhanced the feed intake [28]. The variations
observed could be due to the supplement dosage. Undoubtedly, sustained egg production
at more than 60 weeks of age, suggests that future research directions should focus on the
dynamics of calcium requirements for laying hens throughout the egg production cycle
and the underlying physiology that supports better laying rates and egg quality.
Egg quality traits, including the eggshell strength and albumen quality, are key in-
dicators for consumers’ perceptions and demand for table eggs. Further analysis in the
study showed that a dietary 50 µg/kg 25-OHD3 and 2000 IU/kg VD3 addition caused
a remarkable increase in eggshell strength and thick albumen height. This corroborates
the previous findings that various sources of vitamin D, VD3 , 25-OHD3 , and 1,25(OH)2 D3
caused a significant increase in the Haugh unit of Hy-Line W-36 laying hens at 80 weeks
of age [13]. In addition, partial or complete replacement of VD3 with 25-OHD3 in diets
enhanced the eggshell quality and reduced the rate of broken eggs [29], whereas the in-
clusion of 25-OHD3 in a basal diet containing VD3 improved the eggshell thickness [28].
Eggshell brightness and eggshell weight were improved with dietary 25-OHD3 [27]. An-
other study reported that a dietary 25-OHD3 addition improved the eggshell thickness
with a high stocking density of laying hens. Supplementation of 25-OHD as a substitute
to VD3 in the diets of laying hens (from 1–70 weeks) enhanced the eggshell thickness at
60 weeks of age but had no effect on other egg quality traits [28]. Another study showed
that the dietary addition of different isoforms of vitamin D had no effect on the egg quality
of Lohmann laying hens [30]. These results suggest that dietary supplementation with
25-OHD3 has positive effects on the Haugh unit, eggshell strength, and eggshell thickness
compared with a VD3 addition. However, varying results across the research studies could
be attributed to the age of laying hens, dosage of the supplement, and viability period of
the functional ingredients.
Blood biochemical indices are used as indicators to evaluate normal body functions
and physiology. In the current study, the dietary 25-OHD3 addition exerted significant
effects on the activities and levels of biochemical markers, such as ALT, ALB, and ALP,
while reducing the level of T-BIL. ALP is an enzyme that is primarily involved in the
deposition of Ca and P and is strongly linked with the rate at which skeletal mineralization
occurs in birds [31]. The reduced content of ALP could be accrued to circulating levels of
25-OHD3 , which aid Ca metabolism and utilization, preventing bone disorders that may
increase its activity. The increased content of ALB could explain the efficient transport
of the minerals and hormones observable in this study, which accounts for the positive
effects on the physiology of the laying hens. In our study, Ca levels in serum in the 50
µg/kg 25-OHD3 + 2000 IU/kg VD3 group and serum P levels in the 50 µg/kg 25-OHD3 +
4000 IU/kg VD3 group enhanced significantly. This corroborates with a previous report
that the dietary addition of different isoforms of VD3 increased the utilization of Ca and
P in laying hens [30]. Also, a study showed that a dietary 69 µg/kg 25-OHD3 addition
increased the content of Ca in serum but had no effect on the P content [32]. However, a
long-term deficiency or low content of VD3 in the diet led to a drastic decline in the Ca
and P contents in the serum of laying hens, and these variations were notable for various
genotypes [33]. The contents of Ca and P in serum reflect the nutritional status of these
minerals in the body. Therefore, 25-OHD3 improves Ca and P retention and absorption,
which will facilitate the absorption and utilization of these minerals compared with VD3 .
Also, the homeostasis of Ca content in the body is achieved through intestinal ab-
sorption, renal excretion, Ca deposition into the eggshell, and bone metabolism, which
Animals 2024, 14, 878 12 of 15

are jointly regulated by PTH, CT, VD3 , and sex hormones [34]. The critical value of the
Ca level in enhancing reproductive function in aged laying hens was reported [35]. In
the current study, the addition of 50 µg/kg 25-OHD3 and 2000 IU/kg VD3 in the diet
caused an increase in sex hormones (FSH and progesterone), PTH level, and 25-OHD3
level, while levels of CORT, ET, BGP, and CT in the 50 µg/kg 25-OHD3 + 4000 IU/kg VD3
groups reduced significantly. Similar results previously reported that a dietary addition
of 69 µg/kg 25-OHD3 reduced COR, the lipopolysaccharide level in the serum of laying
hens [27]. Nevertheless, a dietary 25-OHD3 addition had no effect on the serum level of
PTH in broiler birds [32]. Additionally, one of the key reliable indicators of vitamin D
status in the body of an animal is the measurement level of 25-OHD3 in the circulating
blood. Preceding studies showed that dietary 25-OHD3 supplementation enhanced the
25-OHD3 circulation level in the blood and promoted bone structure development for
mineral deposition and bone quality during the laying period [19,27,32]. In another study,
increased levels of 25-OHD3 in the serum of laying hens due to dietary treatments were
notable for various breeds of chickens [33]. The positive effects of high circulating 25-OHD3
are accrued to its capacity to supply a sufficient amount of 25-OHD3 compared to regular
vitamin D supplementation. The aforementioned findings are suggestive of adopting
25-OHD3 as a substitute for VD3 in the diet of laying hens during the late-production cycle.
Moreover, the antioxidant capacity and liver function of laying hens declined signif-
icantly during the late-production phase. In one study, increased AST activity, elevated
MDA, a lipid peroxidation product, and decreased activities of CAT, GSH, GSH-Px, and
SOD were observed in aged laying hens [36]. These effects imply reduced antioxidant status
and tissue/organ damage due to the higher activity of AST. Higher activities of antioxidant
enzymes, including SOD, CAT, and GSH-Px, inactivate the ROS; the reason may be that
antioxidant enzymes have unique ways to remove excessive produced free radicals [37,38].
These effects are linked with enhancing a balance between oxidant and antioxidant systems
for stable physiology and animal health. Our findings indicated the potency of dietary
50 µg/kg 25-OHD3 and 2000 IU/kg VD3 supplementation on increased antioxidant capac-
ity and scavenging ability of free radicals compared to the control group. This is consistent
with a previous study that demonstrated the antioxidant effect of dietary 25-OHD3 . In one
study with broiler birds fed diets with low Ca and P levels, dietary 25-OHD3 prevented
oxidative stress-induced osteoporosis [32]. Also, oxidative stress (induced by restricted
feeding [39]), high stocking density [40], and exposure to lipopolysaccharide challenge
in broilers and laying hens, respectively, were alleviated with dietary 25-OHD3 [14]. In
addition, 25-OHD3 increased the activities of antioxidant enzymes in both serum and tissue
(jejunum), maintained intestinal barrier function, and caused a reduction in the levels
of proinflammatory cytokines in laying hens and broiler birds [32,40]. Thus, a dietary
25-OHD3 addition improves the antioxidant function in birds via the upregulation of
antioxidant enzymes and inhibition of oxidants.
A relationship exists between antioxidant status and bone health. Caged laying hens
are often predisposed to osteoporosis and other bone problems due to oxidative stress [41],
and a strong correlation between high levels of oxidative stress and osteoporosis has been
reported [42]. Due to an imbalance in the oxidant–antioxidant system, reactive oxygen
species can cause osteocyte death and disturb bone formation. The resultant Ca deficiency
could lead to a decrease in the activity of antioxidant enzymes such as SOD [43]. Therefore,
it is plausible that dietary supplementation of 25-OHD3 can augment mineralization and
bone quality via an improved antioxidant defense system.
Furthermore, the small intestine aids Ca absorption; the transcellular and paracellular
Ca transport channels are most active in the duodenum and jejunum, especially in birds [44].
Earlier studies demonstrated that healthy intestinal conditions could promote the absorp-
tion of nutrients because there were larger absorption areas and a higher turnover rate of
epithelial cells [45]. Nevertheless, the functional role of intestinal morphology often de-
clines with the age of laying hens, but nutritional interventions can modulate these changes.
Our present findings show that the dietary supplementation of 50 µg/kg 25-OHD3 and
Animals 2024, 14, 878 13 of 15

2000 IU/kg VD3 increased villi height and the villi-height-to-crypt-depth ratio compared
to the VD3 group. This is in agreement with previous reports, which showed that dietary
25-OHD3 improved the expression of intestinal barrier-related genes, thus improving in-
testinal integrity [40]. In one study with aged laying hens, combined treatment of biscuit
meal and a by-product of modified olive oil maintained intestinal morphology, improved
digestive function, and increased egg production, thus counteracting the adverse effects of
age on intestine morphology [46]. Therefore, we deduce that compared with VD3 , 25-OHD3
may improve performance, egg quality, circulation of hormones, and mineral contents via
an enhancement effect on intestinal morphology for better nutrient utilization.
A further evaluation of bone quality showed that a dietary 25-OHD3 addition en-
hanced the arrangement of bone trabeculae tightly, the number and area of trabeculae,
and tibia strength. The findings are suggestive of increased bone connectivity, improved
bone trabecular structure, and a higher resistance to fracture. Similarly, a dietary 25-OHD3
addition exerted a positive effect on trabecular bone structure [19], tibia strength, and
structure (dense cortical bone structure), as the medullary cavity was filled with numerous
trabeculae [27]. Interestingly, a decline in the bone quality (bone ash, bone mineral contents,
bone density, bone breaking strength, and stiffness) of broiler birds in low Ca and P level
diets was reversed with the dietary 25-OHD3 addition [32]. Another study reported that a
56 µg/kg 25-OHD3 addition in the diets of pullets caused an improvement in tibia quality
during the laying period [47]. Whereas the long-term dietary supplementation of 25-OHD3
exerted a positive effect on bone strength at 72 weeks of age but not at an earlier age [28],
an indication that the duration of supplement 25-OHD3 in the diets of hens may play a key
role in the observable effects. These results are indicative of the crucial roles of Ca and P in
bone development and bone health status. It has been previously reported that a loss of
trabecular bone could negatively affect bone strength [48]. Also, the capacity of 25-OHD3
to reduce bone deformation is due to its involvement in maintaining Ca and P homeostasis,
which promotes bone mineralization. Thus, the increased bone strength might be linked to
improved bone trabecular structure. Hence, a dietary 25-OHD3 addition can be used to
improve bone fracture resistance in aged laying hens under the cage system and decrease
the incidence of bone disorders.

5. Conclusions
In summary, dietary supplementation of 25-OHD3 maintained the jejunal villi mor-
phology, increased the concentration of circulating 25-OHD3 , sex hormones, Ca regulatory
cues, and increased Ca absorption. These positive effects culminated in enhanced laying
performance, egg quality, and bone health of laying hens during the late-production phase.
Hence, it may be inferred that the superiority of 25-OHD3 over VD3 indicates that it has
the potential to prevent age-induced osteoporosis in laying hens, thereby contributing to
enhanced performance, egg quality, and blood biochemical parameters.

Author Contributions: Data curation, S.G.; methodology, S.G., K.Q. and J.Z.; writing—original draft,
S.G.; project administration, S.W.; supervision, S.W. and J.W.; writing—review and editing, S.W. and
X.Q.; formal analysis, H.Z.; investigation, X.Q. All authors have read and agreed to the published
version of the manuscript.
Funding: This study was supported by Beijing Innovation Consortium of Agriculture Research Sys-
tem (BJJQ-G13), National Natural Science Foundation of China (32272907), and Agricultural Science
and Technology Innovation Program (ASTIP) of the Chinese Academy of Agricultural Sciences.
Institutional Review Board Statement: The experimental protocol used in this study was approved
by the Animal Care and Use Committee (IFR-CAAS-20220401) of the Institute of Feed Research of
the Chinese Academy of Agriculture Science.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained in this article.
Conflicts of Interest: The authors declare no conflicts of interest.
Animals 2024, 14, 878 14 of 15

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