p-ISSN 2615-787X e-ISSN 2615-790X YUSUF ET AL.

/ Tropical Animal Tropical Animal

Science Journal Science Journal, December 2023, 46(4):461-470
46(4):461-470
Accredited by Directorate General of Higher Education, Research, DOI: https://doi.org/10.5398/tasj.2023.46.4.461
and Technology, Republic of Indonesia, No. 225/E/KPT/2022 Available online at https://journal.ipb.ac.id/index.php/tasj

Performance, Egg Quality, Bone Health, and Immunity Assessments of Lohmann

Laying Hens Supplemented with Vitamin D3 in the Diet

G. M. Yusufa, Sumiatib,*, R. Mutiab, W. W. Wardanic, I. Akbarc, & N. D. S. Putric

a
Graduate School of Nutrition and Feed Science, Faculty of Animal Science, IPB University
b
Department of Nutrition and Feed Technology, Faculty of Animal Science, IPB University
Jalan Agatis, Kampus IPB Dramaga, Bogor 16680, West Java, Indonesia
c
Nutricell R&D, Cibis Nine 12th Floor Unit G1, Jakarta 12560, Indonesia
*Corresponding author: [email protected]
(Received 01-07-2023; Revised 25-09-2023; Accepted 26-09-2023)

ABSTRACT

Bone health in laying hens is associated with production performance and physical egg quality.
This study aimed to evaluate the effects of a diet supplemented with Nutricell Eggstra (vitamin
D3 enriched with canthaxanthin) on the production performance, egg quality, bone mineral, and
immunity of laying hens. Herein, 150 Lohmann laying hens were divided into three treatments
with five replicates. The treatment diets were: T1= diet without Nutricell Eggstra supplementation
(control), T2= diet supplemented with 1.00 kg/ton Nutricell Eggstra, and T3= diet supplemented with
1.50 kg/ton Nutricell Eggstra. Compared to the control diet, supplementation of Nutricell Eggstra at
doses of 1.00 kg/ton and 1.50 kg/ton significantly increased (p<0.05) hen day production, egg mass,
egg index, yolk index, albumen weight, yolk color, eggshell thickness, eggshell strength, vitamin
D3 content in egg yolk, heterophil, and heterophil/lymphocyte ratio. Supplementation of Nutricell
Eggstra at the studied doses significantly decreased (p<0.05) the feed conversion ratio, yolk weight,
eggshell porosity, and lymphocyte content. Nutricell Eggstra supplementation did not affect feed
consumption, egg weight, albumen index, albumen weight, eggshell weight, Haugh unit, and tibia
bone minerals. In conclusion, supplementation of 1.00 kg/ton Nutricell Eggstra yielded the best
performance for Lohmann laying hens aged 36-39 weeks, while supplementation of 1.50 kg/ton
Nutricell Eggstra produced the best quality eggs.

Keywords: bone; egg quality; immunity; Lohmann hens; vitamin D3

INTRODUCTION ence economic losses owing to the reduced quality of

their bones and eggshells.
Owing to the exponential increase in the human Ensuring a balance between the medullary bone
population, there has been an exponential increase in and structural formation is crucial for bone quality
the demand for table eggs worldwide (El-Sabrout et and egg production (Adhikari et al., 2020). During egg-
al., 2022). Globally, eggs have become one of the main shell synthesis, blood Ca is rapidly mobilized, and its
animal protein sources for society, based on data from level decreases stimulating the secretion of parathyroid
the Food and Agriculture Organization of the United hormones (PTH), which promotes bone resorption to
Nations (2022), accounting for 6% of the total hen egg maintain Ca homeostasis (Sinclair-Black et al., 2023).
production in the world, Indonesia ranks fourth in When bone resorption exceeds formation, the bone ma-
hen egg production after China, India, and the United trix becomes fragile and prone to fracture (Duran et al.,
States of America. According to the Central Bureau 2018). Improving feed nutrition is a possible solution.
of Statistics, the demand for hen eggs in Indonesia The feed nutrition strategy focuses on maintaining bone
increases annually. The demands for hen eggs in 2020, health and the physical quality of eggs and improves the
2021, and 2022 were 4,895,998, 5,066,543, and 5,310,278 production performance and nutritional content of eggs
tons, respectively. High egg-producing laying hens suf- for human consumption (El-Sabrout et al., 2022).
fer from osteoporosis during the laying period because Vitamin D is an essential fat-soluble vitamin for Ca
the medullary and structural bones are progressively and phosphorus (P) metabolism and maintaining bone
absorbed to supply calcium (Ca) for eggshell formation. integrity in chickens (Adhikari et al., 2020). Vitamin D
This has become a severe economic and welfare problem also plays a role in Ca and P absorption and homeosta-
for the poultry industry (Chen et al., 2020). Attia et al. sis, PTH regulation, bone mineralization and mobiliza-
(2020) reported that the production performance and tion, and the control of bone abnormalities (Garcia et
eggshell quality of laying hens were significantly influ- al., 2013). Two classes of vitamin D physiologically
enced by their bone qualities, and farmers may experi- influence hormones: ergocalciferol (vitamin D2) and cho-

Copyright © 2023 by Authors, published by Tropical Animal Science Journal. December 2023 461
This is an open-access article distributed under the CC BY-SA 4.0 License
(https://creativecommons.org/licenses/by/4.0/).
 YUSUF ET AL. / Tropical Animal Science Journal 46(4):461-470

lecalciferol (vitamin D3). Vitamin D2 and vitamin D3 are trol), T2= diet supplemented with 1.00 kg/ton Nutricell
hydroxylated in the liver by 25-hydroxylase to become Eggstra, and T3= diet supplemented with 1.50 kg/ton
25OHD2 / 25OHD3, which is then hydroxylated in the kid- Nutricell Eggstra. The birds were kept in individual
ney by 1α-hydroxylase into the active form 1,25(OH)2D3 cages equipped with a feeding trough and nipple drinker.
(Holick & Chen, 2008). The active form synergizes with The temperature generated during the research was
PTH, which involves intestinal Ca absorption, skeletal 25.20-31.65 °C with humidity 72.65%-96.64%. Birds were
Ca deposition, and Ca resorption from bone tissue when offered feed twice a day (07.00 and 16.00 h) with a mash
plasma Ca levels are low (Saunders-Blades & Korver, diet based on the nutritional requirements of Lohmann
2014). laying hens (Lohmann Management Guide, 2019) (Table
Supplementation of vitamin D3 in the laying hen diet 2) 110 g/bird/day. The laying hens were reared in an open
has been reported to increase egg production and physi- house system with a concrete floor and rice husk litter.
cal quality (Geng et al., 2018), as well as improve the tibia
bone quality of laying hens during the egg-laying period Performance of Laying Hens
(Li et al., 2021). The vitamin D3 in the feed is absorbed into
the bloodstream and transferred to the egg yolk through The performance of laying hens was measured at 36-
the ovaries. The increase in the vitamin D3 content of 39 weeks of age. According to the Lohmann Management
eggs is in line with the increase in vitamin D3 in the diet Guide (2019), this age range is classified as the egg-laying
(Yao et al., 2013). Visual appearance, especially color, is period Phase 1. The diet for Phase 1 was designed to
vital in food selection, including eggs. Using carotenoids meet the maximum egg mass production requirement.
to pigment animal products to enhance egg yolk color is Supplementation with vitamin D3 during the rearing
widely accepted in the poultry industry. Canthaxanthin is and laying stages could promote bone development and
a carotenoid commonly used in diets, contributing to the protect against bone loss during egg production, which
orange-red color of egg yolk. This pigment also has the may benefit the birds during the later laying period.
potential as an antioxidant for birds and their derivative Feed consumption was recorded daily and calculated
products (Faruk et al., 2018). as the difference between the amount of feed given and
The efficiency of vitamin D3 supplementation de- the remaining feed. The feed conversion ratio (FCR) was
pends on several factors, such as dose, age, production obtained by calculating the ratio between the amount
phase, and duration of the study. Information regarding of feed consumed and the mass of eggs produced. Hen
vitamin D3 enriched with canthaxanthin has not been day production (HDP) was calculated as the ratio of
widely studied. Hence, we hypothesized that laying hens the number of eggs produced to the number of laying
require an appropriate vitamin D3 content during the hens. Egg weight was calculated based on the results of
production stage, and supplementation with Nutricell daily egg weighing during rearing, and egg mass was
Eggstra as a vitamin D3 source enriched with canthaxan- calculated by multiplying egg production by egg weight.
thin in the diet may increase the production, egg quality,
bone minerals, and profits for laying hen farmers. This Egg Quality
study evaluated the effects of Nutricell Eggstra supple-
mentation in the diet on performance, egg quality, bone In total, 90 eggs (six eggs per replicate) were used for
mineral content, and immunity of Lohmann laying hens physical egg quality measurements. The measured physi-
aged 36-39 weeks. cal quality variables included the egg index, percentage
of albumen weight, yolk weight, eggshell weight, Haugh
MATERIALS AND METHODS unit, yolk color, eggshell color, eggshell thickness, egg-
shell strength, and eggshell porosity. An electronic digital
Ethical Approval balance was used to measure the egg, yolk, eggshell, and
albumen weights. Albumen and eggshell thicknesses
All experimental procedures were approved by the were measured using a digital micrometer. The Haugh
Animal Ethics Committee of IPB University according to unit (HU) was calculated using the Haugh formula
the Guidelines for the Care and Use of Animals (Number: (Haugh, 1937), HU= 100 log [H + 7.57 − 1.7W0.37], where H
041-2023 IPB). was the albumen thickness (mm) and W was the weight
of the entire egg (g). The shape index was calculated as
Animal and Diets the ratio of the egg width to the egg length. Eggshell
strength was measured using an RH-DW200 Eggshell
This study was conducted at the Department of Strength Tester (Guangzhou RunHu Instruments Co.,
Nutrition and Feed Technology, Faculty of Animal Ltd.). The eggshell color and eggshell porosity were as-
Science, IPB University, between October and December
2022. This research used 150 Lohmann laying hens aged Table 1. Product specification of Nutricell Eggstra (kg)
36–39 weeks, which were distributed according to a
completely randomized design into three experimental Material Amount Unit
treatments, with five replicates of ± 10 birds each. The Vitamin D3 3,500,000 IU
supplement product used was Nutricell Eggstra, which Canthaxanthin 5.5 g
is vitamin D3-enriched canthaxanthin from PT. Nutricell Antioxidant* 1.0 g
Pacific (Table 1). The treatments were as follows: T1= Note: *BHA (butylated hydroxyanisole), BHT (butylated hydroxytolu-
diet without Nutricell Eggstra supplementation (con- ene), ethoxyquin.

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Table 2. The composition and nutritional value of the experi- hematocrit, hemoglobin, and differential leukocyte
mental diets counts. The numbers of erythrocytes and leukocytes
Treatments were calculated using the Neubauer counting chamber
Items method and observed under a microscope. The number
T1 T2 T3
Ingredients
of red and white blood cells were calculated using
Hayem’s and Reez-Ecker’s diluents. Hemoglobin
Corn (%) 55.50 55.50 55.50
was analyzed using the Sahli method with HCl (0.1
Wheat bran (%) 2.50 2.50 2.50
N). Differential leukocyte counts were performed by
Soybean meal (%) 18.00 18.00 18.00
preparing smears with May Grunwald-Giemsa staining
Fish meal (%) 7.00 7.00 7.00
(Schalm, 1961) and observed using a microscope.
Corn gluten meal (%) 4.60 4.60 4.60
Monocytes lymphocytes, heterophils, basophils, and
Crude palm oil (%) 2.00 2.00 2.00 eosinophils were differentiated. The hematocrit value
CaCO3 (%) 8.50 8.50 8.50 was measured using a microcapillary tube filled with
Dicalcium phosphate (%) 1.00 1.00 1.00 blood until it reached 4/5. The end of the tube was
NaCl (%) 0.30 0.30 0.30 plugged with a crest seal, the tube was centrifuged for
Premix (%)3 0.50 0.50 0.50 3 min, and the hematocrit value was measured with a
DL-methionine (%) 0.10 0.10 0.10 microhematocrit tube reader.
Total 100 100 100
Supplementation of Nutricell 0.00 1.00 1.50 Tibia Bone Minerals Analysis
Eggstra (kg/ton)
Nutrients content Laying hens from each treatment were slaughtered
Dry matter (%)1 89.58 89.58 89.58 according to the Islamic method at the end of the study
Metabolizable energy (kcal/kg)2 2,855 2,855 2,855 to collect the tibia. The meat and tibia were soaked in
Gross energy (kcal/kg)1 3,608 3,608 3,608 hot water, cleaned, and analyzed for mineral content,
Crude protein (%)2 18.90 18.90 18.90 namely Ca, P, and zinc (Zn) using the AOAC method
Crude fat (%)1 3.01 3.01 3.01 (2005). Dry and mashed samples were weighed in a
Crude fiber (%)1 4.96 4.96 4.96 porcelain dish ±2–10 g, then put into a furnace for 4 h
Calcium (%)1 3.67 3.67 3.67 at 550 °C. Thereafter, 10 mL of 3 M HCl was added to
Total phosphorus (%)1 1.49 1.49 1.49 the samples and heated on a hotplate for 10 min. The
Available phosphorus (%)2 0.60 0.60 0.60 heated solution was then added to 100 mL of distilled
Lysine (%)2 1.10 1.10 1.10 water in a measuring flask and filtered. The responses
Digestible lysine (%)2 0.99 0.99 0.99 of Ca and Zn were measured using an atomic ab-
Methionine (%)2 0.50 0.50 0.50
sorption spectrophotometer, where P was measured
spectrophotometrically.
Digestible methionine (%)2 0.50 0.50 0.50
Methionine + cysteine (%)2 0.90 0.90 0.90
Immune Organs
Digestible methionine + cysteine 0.70 0.70 0.70
(%)2
Immune organs, such as the spleen, thymus, and
Note: ¹Analysis results from PAU Laboratory IPB University 2023.
²Based on formulation calculations using least cost formulation. bursa of Fabricius were separated and weighed to cal-
3
5 kg premix contains: vitamin A 12.500 IU; vitamin D3 2.500 IU; culate the weight percentages. The relative weight of the
vitamin E 10.000 mg; vitamin K3 2.000; vitamin B1 2.000 mg; vi- immune organs (thymus, spleen, and bursa of Fabricius)
tamin B2 4.000 mg; vitamin B6 1.000 mg; vitamin B12 12,000 mcg;
vitamin C 40.000 mg; niacin 40.000 mg; biotin 200 mg; l-arginine
were computed using the body weight at slaughter (Lan
10.000 mg; l-threonine 15,000 mg; Dl-methionine 5.000 mg; l-ly- et al., 2017).
sine 125.000 mg; folic acid 500 mg; ferrous 30.000 mg; Mn 60.000
mg; Cu 5.000 mg; iodide 200 mg; Se 200 mg; and Co 200 mg. Statistical Analysis
T1= diet without Nutricell Eggstra supplementation; T2= diet
supplemented with 1.00 kg/ton Nutricell Eggstra; T3= diet supple-
mented with 1.50 kg/ton Nutricell Eggstra. Data were statistically analyzed using various tests.
When a treatment yielded a significant result (p<0.05),
the Duncan’s multiple range test was used to determine
sessed using the grading and scoring systems provided significant differences among the mean values. Analysis
by Nutricell Eggs-Pert, a tool from PT Nutricell Pacific. of variance was performed using the SPSS Statistics soft-
The yolk color index was determined using the yolk ware version 25 (2017).
color fan scale. The measured variables of egg chemical
quality included vitamin D3 content in the egg yolk. The RESULTS
vitamin D3 content was analyzed using liquid chroma-
tography mass spectrometry (Shimadzu LCMS 8045 Performance of Laying Hens
2022).
Nutricell Eggstra supplementation in the diet of
Blood Profile Analysis Lohmann laying hens aged 36–39 weeks showed a
significant effect (p<0.05) on HDP, egg mass produc-
Blood hematology profile analysis included the tion, and FCR, but it had no significant effect on feed
number of erythrocytes and leukocytes, as well as the consumption and egg weight (Table 3). Our results

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displayed an increase in HDP by 12.44% (T2) and 9.01% egg shape index, yolk index, percentage of albumen
(T3) compared to the control (T1). Laying hens whose and egg yolk, yolk color, eggshell thickness, eggshell
diets were supplemented with Nutricell Eggstra had a strength, eggshell porosity, and vitamin D3 content in
weekly increasing HDP value, whereas those that were the egg yolk. However, it did not show a significant
only fed a control diet had a fluctuating HDP in line effect (p>0.05) on the albumen index, percentage of egg-
with egg mass production. While the FCR fluctuated shell, HU, and eggshell color (Table 4). Eggshell color,
throughout the experimental period, treatment with thickness, and strength were correlated with eggshell
Nutricell Eggstra supplementation at a 1.00 kg/ton dose exterior quality in this experiment. Nutricell Eggstra
had the lowest FCR compared to other treatments. At supplementation in the Lohmann laying hens diet
36 and 38 weeks, the FCR peaked for all the treatments. resulted in an improved eggshell quality. According to
At 37 and 39 weeks, the FCR gradually decreased, but the eggshell porosity score (%), its eggshell quality was
the control diet treated birds maintained a high FCR classified as good (score 2) compared to the control diet,
throughout. Nutricell Eggstra supplementation at 1.00 which scored an average (score 3). The level of Nutricell
and 1.50 kg/ton did not have different FCRs, but both Eggstra supplementation was correlated with the vi-
were significantly lower than the control diet. These re- tamin D3 content of the egg yolk, as an increase in the
sults indicate that a Nutricell Eggstra supplemented diet supplement’s level significantly increased the Vitamin
had the highest feed efficiency, with a decrease in FCR D3 content.
of 11.61 (T2) and 9.37% (T3) compared to the control
diet. The best income over feed cost (IOFC) value was Blood Profile
achieved in laying hens supplemented with Nutricell
Eggstra at as much as 1.00 kg/ton with a profit of IDR The effects of Nutricell Eggstra supplementation on
26,257.69/bird. blood profiles in the Lohmann laying hens diet at 36–39
weeks of age are shown in Table 5. The addition of the
Egg Quality Nutricell Eggstra supplement significantly (p<0.05)
decreased the percentage of lymphocytes and increased
Laying hens supplemented with Nutricell Eggstra the rate of heterophiles and the ratio of heterophiles/
in their diets showed significantly increased (p<0.05) lymphocytes (H/L). However, no significant differences

Table 3. Performance of Lohmann laying hens aged 36–39 weeks supplemented with Nutricell Eggstra (vitamin D3 enriched with
canthaxanthin)

Treatments
Variables
T1 T2 T3
Feed consumption (g/bird/day) 105.46 ± 1.26 105.77 ± 1.22 105.15 ± 1.03
Hen day production (%) 83.86 ± 1.80c 94.29 ± 0.56a 91.42 ± 0.71b
Egg weight (g) 56.29 ± 1.35 56.53 ± 0.22 56.71 ± 1.17
Egg mass (g/bird) 47.16 ± 1.85b 53.30 ± 0.27a 51.82 ± 1.27a
Feed conversion ratio 2.24 ± 0.08a 1.98 ± 0.30b 2.03 ± 0.30b
Income over feed cost (IDR/bird)* 6,845.09 26,257.69 24,476.04
Note: T1= diet without Nutricell Eggstra supplementation, T2= diet supplemented with 1.00 kg/ton Nutricell Eggstra; T3= diet supplemented with 1.50
kg/ton Nutricell Eggstra. Means in the same row with different superscripts are significantly different (p<0.05).
*Normal egg price on Farm IDR 22.500.00 per kg, vitamin D enriched egg price on Farm IDR 33,750.00 per kg and feed price IDR 7.740.00.

Table 4. Egg quality of Lohmann laying hens aged 36–39 weeks supplemented with Nutricell Eggstra (vitamin D3 enriched with
canthaxanthin)
Treatments
Variables
T1 T2 T3
Shape index 0.78 ± 0.00ab 0.77 ± 0.00b 0.79 ± 0.00a
Albumen index 0.13 ± 0.00 0.14 ± 0.01 0.13 ± 0.00
Yolk index 0.45 ± 0.00b 0.47 ± 0.00a 0.48 ± 0.00a
Albumen (%) 59.48 ± 1.39b 61.66 ± 0.54a 62.17 ± 0.71a
Yolk (%) 29.74 ± 1.64a 26.99 ± 0.64b 26.26 ± 0.45b
Shell (%) 10.79 ± 0.67 11.37 ± 0.27 11.54 ± 0.45
Haugh unit 94.25 ± 0.67 95.80 ± 1.67 95.14 ± 1.33
Yolk color 6.40 ± 0.54c 13.40 ± 0.54b 14.80 ± 0.44a
Eggshell color 13.20 ± 0.83 13.20 ± 0.83 13.60 ± 0.54
Eggshell thickness (mm) 0.35 ± 0.01b 0.38 ± 0.01a 0.38 ± 0.01a
Eggshell strength (kgf) 2.60 ± 0.30b 2.90 ± 0.07a 3.01 ± 0.19a
Eggshell porosity 3.00 ± 0.00a 2.40 ± 0.54b 2.00 ± 0.00b
Vitamin D3 content in egg yolk (mcg/100 g) 0.00 ± 0.00c 4.37 ± 0.72b 7.11 ± 1.23a
Note: T1= diet without Nutricell Eggstra supplementation, T2= diet supplemented with 1.00 kg/ton Nutricell Eggstra; T3= diet supplemented with 1.50
kg/ton Nutricell Eggstra. Means in the same row with different superscripts differ significantly (p<0.05).

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(p>0.05) were observed in hemoglobin, hematocrit, 36–39 weeks, which was 95.30%–95.00% (Lohmann
erythrocytes, leukocytes, basophils, eosinophils, and Management Guide, 2019). Setiyaningsih et al. (2023)
monocytes. reported that the combination of 25(OH)D3 and vitamin
C contained in Nutricell HyC® can increase HDP in
Tibia Bone Minerals broiler breeders at the age of 34-46 weeks. According to
Geng et al. (2018), vitamin D3 supplementation in lay-
Supplementation of Nutricell Eggstra in the diet ing hens can increase the levels of follicle-stimulating
of Lohmann laying hens aged 36–39 weeks showed no and luteinizing hormone or induce a feedback effect
significant effect (p>0.05) on the content of tibia minerals on hypothalamic and pituitary hormone secretion,
such as Ca, P, and Zn (Table 6). which positively affects follicle development. This sup-
ports the observed positive effect of vitamin D3 on the
Immune Organs performance of laying hens in association with estrogen
hormones. However, various studies have shown that
Supplementation with Nutricell Eggstra in the diet the use of vitamin D3 in laying hen diets does not affect
of Lohmann laying hens aged 36-39 weeks had no sig- egg production (Nascimento et al., 2014; Adhikari et al.,
nificant effect on the average weight and percentage of 2020).
immune organs (Table 7). The average egg weight in this study was still
lower than the standard egg weight of Lohmann laying
DISCUSSION hens aged 36-39 weeks, with a weight of 61.60-62.30
g (Lohmann Management Guide, 2019). According to
Performance of Laying Hens Chen et al. (2020), there was no significant difference
in egg weight, which could be related to the age of the
Egg production can be influenced by several factors birds and the duration of vitamin D3 supplementation.
such as strain, diet nutrients, health and rearing man- Other factors affecting egg weight include genetics,
agement, age at first laying, and peak egg production. body size, climate, egg production, proteins, and amino
The HDP was in line with Geng et al. (2018), vitamin acids (especially methionine), which are essential in con-
D3 supplementation of 500–3000 IU/kg can produce trolling egg size (Leeson & Summers, 2005). This is also
an HDP of 90.30%–94.03%, but this was still lower supported by Pakpahan et al. (2023), who found that
than the standard HDP of Lohmann laying hens aged one of the factors affecting egg weight is the nutritional

Table 5. Blood profile of Lohmann laying hens aged 39 weeks supplemented with Nutricell Eggstra (vitamin D3 enriched with
canthaxanthin)

Treatments
Variables
T1 T2 T3
Hemoglobin (g%) 8.68 ± 1.46 9.04 ± 1.45 9.28 ± 0.59
Hematocrit (%) 22.00 ± 2.34 19.80 ± 1.78 21.00 ± 2.00
Erythrocytes (106/mm3) 2.01 ± 0.62 2.53 ± 0.93 2.42 ± 0.74
Leukocytes (103/mm3) 13.72 ± 6.11 12.44 ± 9.20 11.09 ± 5.54
Lymphocytes (%) 55.70 ± 2.16a 51.74 ± 2.96b 52.85 ± 1.39ab
Monocytes (%) 3.06 ± 1.03 2.97 ± 0.91 2.79 ± 1.78
Heterophils (%) 31.31 ± 2.67b 34.56 ± 2.15a 35.13 ± 1.12a
Eosinophils (%) 2.19 ± 0.46 1.98 ± 0.69 1.69 ± 0.53
Basophils (%) 7.73 ± 1.40 8.73 ± 2.16 7.50 ± 1.37
Ratio H/L 0.56 ± 0.07b 0.67 ± 0.07a 0.66 ± 0.03a
Note: T1= diet without Nutricell Eggstra supplementation, T2= diet supplemented with 1.00 kg/ton Nutricell Eggstra; T3= diet supplemented with 1.50
kg/ton Nutricell Eggstra. Means in the same row with different superscripts differ significantly (p<0.05).

Table 6. Tibia bone minerals content of Lohmann laying hens Table 7. Average weight and percentage of immune organs in
aged 39 weeks supplemented with Nutricell Eggstra Lohmann laying hens aged 39 weeks supplemented
(vitamin D3 enriched with canthaxanthin) with Nutricell Eggstra (vitamin D3 enriched with
canthaxanthin)
Treatments
Variables
T1 T2 T3 Treatments
Variables
Ca (%) 39.98 ± 1.13 38.45 ± 1.89 39.72 ± 1.28 T1 T2 T3
P (%) 5.77 ± 0.49 5.12 ± 0.64 5.42 ± 0.45 Thymus (g) 0.08 ± 0.23 0.78 ± 0.33 1.02 ± 0.58
Zn (ppm) 128.02 ± 7.05 127.86 ± 7.08 131.39 ± 8.48 (%) 0.05 ± 0.01 0.04 ± 0.01 0.06 ± 0.03
Note: T1= diet without Nutricell Eggstra supplementation, T2= diet Spleen (g) 2.88 ± 1.26 2.80 ± 1.36 2.02 ± 0.45
supplemented with 1.00 kg/ton Nutricell Eggstra; T3= diet supple- (%) 0.18 ± 0.08 0.17 ± 0.07 0.12 ± 0.02
mented with 1.50 kg/ton Nutricell Eggstra.
Note: T1= diet without Nutricell Eggstra supplementation, T2= diet
supplemented with 1.00 kg/ton Nutricell Eggstra; T3= diet supple-
mented with 1.50 kg/ton Nutricell Eggstra.

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content of the diet. Egg weight in this study did not dif- IOFC. The value of feed consumption for all treatments
fer between treatments because they were fed the same was relatively the same, even though treatment T2 or T3
nutrient content, especially protein and fat. required a higher total feed cost due to the additional
Nutricell Eggstra supplementation in the diet cost of Nutricell Eggstra supplementation. However,
resulted in higher egg mass production than in the this can be offset by egg mass production, which is
control diet. The results were positively correlated higher than the control treatment, such that the differ-
with HDP because egg mass production is affected ence between egg sales and the total diet cost incurred
by egg production, egg weight, and heat stress (Yan during the rearing period increases.
et al., 2022). The laying hens receiving a Nutricell
Eggstra supplemented in the diet had higher egg mass Egg Quality
production than those reported by Wen et al. (2019) that
laying hens received a vitamin D3 supplementation of The egg shape index is related to the shape of the
1681–68348 IU/kg had egg mass production of 50.40– egg, with an ideal value of 0.80 or oval in shape. The
51.80 g/bird. However, a lower egg mass production albumen index in this study was included in quality I,
was observed compared to Geng et al. (2018), which was with a value of 0.134–0.175 (BSN, 2008). According to
as high as 54.55–56.83 g/bird after receiving 500–3000 Obianwuna et al. (2022), protein in the diet can affect
IU/kg of supplemented vitamin D3. Setiyaningsih et al. albumen viscosity. The thicker the albumen, the higher
(2023) reported that combining 25(OH)D3 and vitamin C the albumen index required to maintain quality. The
increased egg mass in broiler breeders at 34-46 weeks of insignificant difference in the albumen index values
age. The improvement in the gut microbial composition in this study was aligned with the constant diet
induced by adding vitamin D3 also plays a role in protein content in each treatment. Nutricell Eggstra
maintaining gut health and improving egg-laying supplementation significantly increased the yolk
performance (Guo et al., 2022). index compared to the control diet, and this value
Nutricell Eggstra supplementation in the diet was included in quality I with a value of 0.458–0.521
resulted in higher feed efficiency when compared to (BSN, 2008). According to Khan et al. (2016), the factors
the control diet. This resulted in laying hens receiving that can affect the yolk index include storage time,
Nutricell Eggstra supplementation have a higher egg temperature, vitelline membrane quality, and nutrition
mass with the same feed consumption. Geng et al. from the diet. The transfer of cholecalciferol to the
(2018) reported that laying hens receiving vitamin D3 egg yolk is highly efficient and responsive to dietary
supplementation of 500-3000 IU/kg had FCR 2.07-2.12, enrichment (Yao et al., 2013). According to Geng et
which was higher than that observed in this study. al. (2018), vitamin D3 is involved in the regulation of
The average FCR in this study follows the standard hormone synthesis and secretion, including that of
FCR for Lohmann layer periods of 2.0–2.1 (Lohmann reproductive hormones, such as estrogen. Adequate
Management Guide, 2019). Vitamin D3 supplementation vitamin D3 levels may promote proper hormonal
in the diet of laying hens can influence physiological signaling, leading to optimal yolk development and
processes that collectively contribute to the improved potentially larger yolk size, contributing to an increased
egg production, bone health, and nutrient utilization, yolk index. Estrogen influences the development of
leading to an improved feed conversion ratio. Attia et the reproductive tract and plays a crucial role in egg
al. (2020) found that vitamin D3 supplementation in the yolk formation by stimulating the liver to produce
diet of laying hens could influence various physiological the yolk precursors vitellogenin and very-low-density
processes that collectively contribute to better egg lipoprotein, which are the primary sources of yolk
production, bone health, and nutrient utilization, protein and lipids, respectively (Schneider, 2016).
leading to improved FCR. An improved microbial The percentages of albumen and eggshells
balance positively impacts gastrointestinal health obtained were higher than those observed in a study
and increases nutrient utilization. Guo et al. (2022) by Wen et al. (2019) (57.60%–57.90% and 9.50%–9.70%,
reported that adequate vitamin D3 supplementation respectively), which used 1681–68348 IU/kg of vitamin
positively affected the intestinal bacterial microbiota. D3 supplement. The percentage of yolk weight obtained
Vitamin D3 increases the composition of gut microbes was higher than those observed in a study by Plaimast
such as Lactobacillales, Lactobacillaceae, Lactobacillus, et al. (2015) (23.75%-25.53%), which used 2000–6000 IU/
and Bacilli, which play an essential role in maintaining kg of vitamin D3 supplementation. Salles et al. (2013)
gut health and optimal egg production. Bacillus spp. reported that vitamin D3 supplementation in the diet
are considered beneficial microbiota and used as feed can increase protein synthesis, which can contribute to
additives and antibiotic alternatives due to the advanced albumen formation, indicating that laying hens do not
feed absorption, which can lead to better growth experience a lack of protein during albumen formation
and production efficiency, and affects FCR positively but correlates with the egg yolk weight, which tends
(Zhen et al., 2018), secret digestive enzymes, produces to be lower. The relationship between vitamin D3
antimicrobial substances and possesses immune- supplementation in the diet of laying hens and a
regulatory and anti-inflammatory effects (Muras et al., decrease in egg yolk weight is not simple and depends
2021). on various factors, including the dosage of vitamin
IOFC is used to determine profit. According to D3, duration of supplementation, and overall diet
Afandi et al. (2020), increasing feed consumption and composition (Chen et al., 2020).
decreasing egg and egg mass production can reduce

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The higher the albumen content, the higher the D3 supplementation of 500–3000 IU/kg. The eggshell
HU value, indicating improved egg quality. The strength and thickness are correlated with an increase of
average HU value in this study was within the normal eggshell strength and thickness in laying hens supple-
range according to the United States Department mented with vitamin D3. The role of vitamin D3 in Ca
of Agriculture Standards (2000), and the eggs were and P metabolism is fundamental for bone development
categorized as AA-quality eggs (HU>72). The absence and eggshell formation (Attia et al., 2020). Increasing the
of a significant difference in the HU values is consistent strength of shells and reducing egg loss are important
with the results of the other studies on the different economic goals in commercial terms.
levels of vitamin D3 supplementation (Plaimast et al., Microbial penetration into eggs can be affected by
2015; Geng et al., 2018; Chen et al., 2020). According to various factors, such as membrane components, eggshell
North & Bell (1990), the HU value is generally more characteristics (porosity and quality), temperature,
indicative of the length of egg storage, and it is not humidity, and storage. Pathogenic bacteria and harmful
affected by the diet, provided the protein and metabolic microorganisms can enter eggs with high porosity (Gole
energy are balanced. The HU value did not differ among et al., 2014). The results of this study are in line with
treatments because the laying hens received a diet with those reported by Setiyaningsih et al. (2023), which also
the same protein and energy content, and the eggs were found that the combination of 25(OH)D3 and vitamin C
stored at the same place and for the same period. contained in Nutricell HyC®️ could significantly decrease
The diet supplemented with 1.50 kg/ton Nutricell the eggshell porosity compared to the control diet in
Eggstra had the highest egg yolk color score compared broiler breeders. Vitamin D3 contained in Nutricell
to other treatments. The results were higher than Eggstra could potentially enhance the absorption of Ca
those of Attia et al. (2020), which used a vitamin D3 and P in the small intestine and promote Ca deposition
supplementation of 800–1200 IU/kg and observed within the bones. Fritts and Waldroup (2003) stated
egg yolk color scores of 7.78–7.87. The color of the that Ca deposition is particularly significant in creating
yolk depends on the source and levels of carotenoids calcium carbonate (CaCO3) during eggshell formation
in the diet (Nabi et al., 2020). Commercial carotenoid within the uterus, which lasts for 20-21 h. Wang et al.
pigments, such as canthaxanthin, are widely accepted (2020) noted that supplementation with vitamin D3 in
and commonly used in the poultry feed industry, and the diet of laying hens increased the levels of serum
Nutricell Eggstra contains 5.50 g/kg of canthaxanthin. vitamin D3 and the presence of carbonic anhydrase
Canthaxanthin is an important carotenoid that is serum. This enzyme plays a critical role in facilitating
efficiently distributed or stored in the yolk (Zhang the deposition of CaCO3 during eggshell formation,
et al., 2011) and is responsible for producing the thereby contributing to the ideal eggshell porosity.
yellow-orange color of the egg yolk. A EFSA FEEDAP The transfer of vitamin D3 from the diet to egg
Panel study (2014) showed that the egg yolk color yolks was highly efficient and responsive to the enrich-
was significantly different when the canthaxanthin ment level in the diet. The results of this study correlate
supplementation varied between 0–60 mg/kg of diet, with those obtained by Yao et al. (2013), in which an
namely 6.38–14.90. These results align with the color of increase in the vitamin D3 content of eggs will be in line
the egg yolks in this study, where an improvement in with the increase in vitamin D3 in the diet. Additionally,
the color of the egg yolks corresponded with an increase vitamin D3 was absorbed into the bloodstream and
in vitamin D3 supplementation and can be associated transferred to egg yolks through the ovaries. Plasma
with the canthaxanthin content in the product. cholecalciferol is precipitated in egg yolk by forming
The main factors that affect eggshell color are a complex with the protein binding to vitamin D. The
genetics, stress, age, and disease (infectious bronchitis) supplementation of Nutricell Eggstra in diets aims to
(Aygun, 2014). In addition to the type of pigment, egg- optimize the productivity of laying hens and improve
shell color can also be affected by the concentration of egg quality by providing added value to products rich
pigments and the structure of the eggshell (Samiullah in vitamin D3. Bio-fortification of animal foods enriches
et al., 2015). Nutricell Eggstra supplementation has food products, where the nutrients of interest are added
the same shell thickness and contrasts with the control through animal feed. Eggs have garnered significant
group with a lower thickness. This aligns with the result attention as a vehicle for vitamin D bio-fortification and
reported by Chen et al. (2020), who used a vitamin D3 enrichment. The economic impact of promoting en-
supplementation of 2760–5520 IU/kg, and obtained riched eggs as an effective food source increases vitamin
a thickness of 0.37–0.39 mm. According to Leeson & D intake, productivity, and profits for farmers.
Summers (2005), the primary nutrients that affect egg-
shell thickness are Ca, P, and vitamin D3. Nascimento Blood Profile
et al. (2014) reported that supplementation of vitamin
D3 increases the percentage and thickness of eggshells. The average hemoglobin level in this study ranged
When the concentration of vitamin D3 in the diet de- from 8.68–9.28 g%, and these results were within the
creases, laying hens respond by reducing the thickness normal range. Laying hens are resistant to internal
of the eggshells produced (Kaur et al., 2013). physiological changes in an effort to maintain a balance
Eggshell strength plays a vital role in determining in the internal body environment. Thus, the hematocrit
egg quality. The results showed lower eggshell strength value tends to decrease during egg laying due to the
than those reported by Geng et al. (2018), which had process of hemodilution, namely an increase in blood
an eggshell strength of 3.23–3.94 kgf using a vitamin plasma volume and erythrocyte mass. Plasma levels

December 2023 467

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return to normal when the last follicle ovulates (Hu et Kerschnitzki et al. (2014), the medullary bone of birds
al., 2021). changes rapidly during the oviposition cycle, and its
Pantaya & Utami (2020) stated that the number mineral content decreases significantly during the shell
of erythrocytes is closely related to hemoglobin and formation process. The average tibia Ca and P mineral
hematocrit levels. Nutricell Eggstra supplementation in contents in this study ranged between 38.45%–39.98%
the diet did not interfere with the average erythrocyte and 5.12%-5.77%, respectively, which was higher than
counts and the birds could metabolize their nutrients that observed by Attia et al. (2020), who supplemented
properly. Laying hens reared at high ambient feed with vitamin doses between 800–1200 IU/kg, name-
temperatures are highly susceptible to infectious ly 31.30%-32.60%, but with a lower P mineral content of
diseases and vitamin D deficiency decreases the cellular 13.50%–14.30%.
immune response in hens. Vitamin D3 supplementation According to Kakhki et al. (2019), maximizing bone
has been shown to have anti-inflammatory activity in mineralization is important because it increases bone
lipopolysaccharide induced avian immune cells (Attia strength and reduces problems related to leg weakness,
et al., 2020), enhancing the innate humoral immunity of morbidity, and mortality in birds. Vitamin D plays an
birds and counteracting inflammatory responses when important role in Ca and P absorption, bone mineral-
exposed to inappropriate immune reactions (Shojadoost ization, and the regulation of PTH (Garcia et al., 2013).
et al., 2015). Leukocytes play an active role in the body’s Vitamin D can regulate collagen maturation because of
defense system. Vitamin D plays an important role in its role as a transcriptional regulator in bones to reduce
enhancing pathogen-specific immune responses, and collagen synthesis. Collagen is a prominent protein in
1,25(OH)2D3 has been shown to increase phagocytosis, bone, and maintaining proper collagen content is impor-
differentiation, and production of antimicrobial tant for building bone health (Duran et al., 2018).
peptides in macrophages (Martens et al., 2020). The The average Zn content in the tibia in this study
study by Saunders-Blades & Korver (2014) showed that ranged between 127.86-131.39 ppm. Groff & Gropper
substituting cholecalciferol with 25OHD3 in the diet (2000) stated that Zn minerals are required for
increased the immune system by increasing leukocyte alkaline phosphatase activity, which plays a role in
bactericidal activity and phagocytic responses. Ca resorption in bone and is responsible for providing
Leukocyte differentials were classified according to inorganic P where required. Schisler & Kienholz (1967)
the type of white blood cells. Lower lymphocyte levels reported that vitamin D deficiency reduces Zn mineral
in laying hens supplemented with vitamin D3 indicate levels in the bones of laying hens. There were no
that the birds were not exposed to infections or disease significant differences in the levels of Ca, P, and Zn in
agents, which is in line with the findings of Hofmann this study, which could be related to the age of the hens
et al. (2020), which stated that a high lymphocyte and the duration of vitamin D3 supplementation.
level indicates that the birds were exposed to infec-
tion. Vitamin D can modulate the function of chicken Immune Organs
lymphocytes by reducing cell proliferation, cytokine
production, and phosphorylation of non-stimulated Immune organs are indicators of poultry health.
lymphocytes (Boodhoo et al., 2016). However, vitamin D The primary lymphoid organs, namely the bursa of
supplementation did not alter the ability of lymphocytes Fabricius and thymus, produce lymphoid cells that
to undergo degranulation (Shojadoost et al., 2015). This function as specific immune systems, including fighting
may be due to the differential effects of vitamin D on the pathogenic bacteria (B lymphoid cells), fighting antigens
functional abilities of T lymphocytes, which can inhibit in cells and destroying cells affected by viruses (T lym-
immunopathology leading to T lymphocyte exhaus- phoid cells) (Owen et al., 2013). At the time of surgery,
tion without inducing general immunosuppression no bursa of Fabricius was observed in any treatment
(Boodhoo et al., 2016). In the body’s first line of defense, group. According to Ko et al. (2018), this occurs because
higher levels of heterophils in laying hens supplement- the bursa of Fabricius is formed from the embryo and
ed with vitamin D3 are associated with the increased can disappear after the birds mature. The bursa of
levels of vitamin D, which increases specificity. Fabricius is then replaced by the other lymphoid organs,
According to Gross & Siegel (1983), stress levels in such as the spleen, thymus, and ceca tonsils. Secondary
poultry can be categorized by the H/L ratio, birds with lymphoid organs, such as the spleen, play an important
H/L ratios of 0.20 (low stress), 0.50 (moderate stress), role in the repair, maturation, and selection of lymphoid
and 0.80 (high stress). The H/L ratios obtained in this cells to deal with specific pathogens.
study indicated that the birds in each treatment expe-
rienced moderate stress levels. This suggests that birds CONCLUSION
experience heat stress owing to environmental tem-
perature (Gross & Siegel., 1983). The increased H/L ratio Supplementing the diet of laying hens with
observed in birds supplemented with vitamin D3 could Nutricell Eggstra can potentially improve egg
be due to the high egg production during the study. production and quality. The best production
performance was observed with 1.00 kg/ton
Tibia Bone Minerals supplementation, while 1.50 kg/ton resulted in better
yolk color, egg yolk vitamin D3 content, and eggshell
The bone quality of laying hens is closely related quality. However, supplementation did not affect
to egg production and eggshell quality. According to the tibia bone mineral content or immunity. Further

468 December 2023

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research is needed to explore the long-term effects of Faruk, M. U., F. F. Roos, & F. Cisneros-Gonzalez. 2018. A meta-
Nutricell Eggstra and Ca supplementation on the bone analysis on the effect of canthaxanthin on egg production
quality and immune response in laying hens. in brown egg layers. Poult. Sci. 97:84-87. https://doi.
org/10.3382/ps/pex236
Food and Agriculture Organization of the United Nation.
CONFLICT OF INTEREST 2022. World Food and Agriculture. FAO Statistics, Rome.
Fritts, C. A. & P. W. Waldroup. 2003. Effects of source and level
W. W. Wardani, I. Akbar, and N. D. S. Putri are of vitamin D on live performance and bone development
employees of PT. Nutricell Pacific, but no conflict of in growing broilers. J. Appl. Poult. Res. 12:45-52. https://
interest could be construed to influence the content of doi.org/10.1093/japr/12.1.45
this study. R. Mutia serves as an editor of the Tropical Garcia, A. F., A. E. Murakami, C. R Duarte, I. C. Rojas, K. P.
Animal Science Journal but has no role in the decision to Picoli, & M. M Puzotti. 2013. Use of vitamin D3 and its
publish this article. metabolites in broiler chicken feed on performance, bone
parameters, meat quality. Asian-Australas. J. Anim. Sci. 26:
408-415. https://doi.org/10.5713/ajas.2012.12455
ACKNOWLEDGEMENT Geng, Y., Q. Ma, Z. Wang, & Y. Guo. 2018. Dietary vitamin
D3 supplementation protects laying hens against
The authors extend their gratitude to PT. Nutricell lipopolysaccharide-induced immunological stress. Nutr.
Pacific for providing the financial support for this Metab. 15:1-14. https://doi.org/10.1186/s12986-018-0293-8
research. Gole, V. C., K. K. Chousalkar, J. R. Roberts, M. Sexton, D.
May, J. Tan, & A. Kiermeier. 2014. Effect of egg washing
REFERENCES and correlation between eggshell characteristics and egg
penetration by various Salmonella Typhimurium strains.
PLoS ONE 9:e90987. https://doi.org/10.1371/journal.
Adhikari, R., D. White, J. D. House, & W. K. Kim. 2020.
pone.0090987
Effects of additional dosage of vitamin D3, vitamin D2,
Groff, J. L. & S. S. Gropper. 2000. The Digestive System:
and 25-hydroxyvitamin D3 on calcium and phosphorus
Mechanism for Nourishing the Body. Advanced Nutrition
utilization, egg quality and bone mineralization in laying
and Human Metabolism. 3rd ed. Wadsworth Thomson
hens. Poult. Sci. 99:364-373. https://doi.org/10.3382/ps/
Learning, Belmont (CA). pp. 24-52.
pez502
Gross, W. B. & H. S. Siegel. 1983. Evaluation of the heterophil/
Afandi, R., B. Hartono, & I. Djunaidi. 2020. The analysis of
lymphocyte ratio as a measure of stress in chickens. Avian
production costs of laying hen farms using semi self-
Dis. 27:972-979. https://doi.org/10.2307/1590198
mixing and total self-mixing feeds in Blitar regency, east
Guo, F., Y. Geng, W. Abbas, W. Zhen, S. Wang, Y. Huang, Y.
Java. Trop. Anim. Sci. J. 43:70-76. https://doi.org/10.5398/
Guo, Q. Ma, & Z. Wang. 2022. Vitamin D3 nutritional
tasj.2020.43.1.70
status affects gut health of Salmonella-challenged laying
AOAC. 2005. Official Methods of Analysis of AOAC
hens. Front. Nutr. 9:888580. https://doi.org/10.3389/
International 18th (Ed). International AOAC, Washington
fnut.2022.888580
DC.
Haugh, R. R. 1937. A new method for determining the quality of
Attia, Y. A., M. A. Al-Harthi, & H. M. Abo El-Maaty. 2020.
an egg. US Poultry Magazine 39:27-49.
Calcium and cholecalciferol levels in late-phase laying
Hofmann, T., S. S. Schmucker, W. Bessei, M. Grashorn, & V.
hens: effects on productive traits, egg quality, blood
Stefanski. 2020. Impact of housing environment on the
biochemistry, and immune responses. Front. Vet. Sci.
immune system in chickens: a review. Animals 10. https://
7:389. https://doi.org/10.3389/fvets.2020.00389
doi.org/10.3390/ani10071138
Aygun, A. 2014. The relationship between eggshell colour and
Hu, J., Y. Xiong, R. S. Gates, & H. W. Cheng. 2021. Perches as
egg quality traits in table eggs. Indian J. Anim. Res. 48:290-
cooling devices for reducing heat stress in caged laying
294. https://doi.org/10.5958/j.0976-0555.48.3.061
hens: a review. Animals (Basel). 11:3026. https://doi.
Boodhoo, N., S. Sharif, & S. Behboudi. 2016. 1α,25(OH)2
org/10.3390/ani11113026
vitamin D3 modulates Avian T lymphocyte functions
Kakhki, R. A. M., T. Heuthorst, A. Wornath-Vanhumbeck, M.
without inducing CTL unresponsiveness. PLoS ONE.
Neijat, & E. Kiarie. 2019. Medullary bone attributes in aged
11:e0150134. https://doi.org/10.1371/journal.pone.0150134
Lohmann LSL-lite layers fed different levels of calcium and
BSN. 2008. Telur Ayam Konsumsi SNI-3926:2008. Badan
top-dressed 25-hydroxy vitamin D3. Canadian J. Anim.
Standardisasi Nasional, Jakarta (ID).
Sci. 99:138-149. https://doi.org/10.1139/cjas-2018-0062
Chen, C., B. Turner, T. J. Applegat, G. Litta, & W. K Kim. 2020.
Kaur, R., B. M. Rathgeber, K. L. Thompson, & J. MacIsaac. 2013.
Role of long-term supplementation of 25-hydroxyvitamin
Uterine fluid proteins and egg quality characteristics for 2
D3 on laying hen bone 3-dimensional structural
commercial and 2 heritage laying hen lines in response to
development. Poult. Sci. 99:5771-5782. https://doi.
manipulation of dietary calcium and vitamin D3. Poult.
org/10.1016/j.psj.2020.06.080
Sci. 92: 2419-2432. https://doi.org/10.3382/ps.2012-02983
Duran, M. R., C. Chen, & W. K. Kim. 2018. Effects of vitamin D
Kerschnitzki, M., T. Zander, P. Zaslansky, P. Fratzl, R. Shahar,
and calcium for the prevention of osteoporosis at various
& W. Wagermaier. 2014. Rapid alterations of avian
stages of life of laying hens-review. Int. J. Poult. Sci. 17:
medullary bone material during the daily egg-laying cycle.
405-409. https://doi.org/10.3923/ijps.2018.405.409
Bone 69:109–117. https://doi.org/10.1016/j.bone.2014.08.019
EFSA FEEDAP Panel (EFSA Panel on Additives and Products
Khan, M. J. A., S. H. Khan, A. Bukhsh, M. I. Abbass, & M.
or Substances used in Animal Feed). 2014. Scientific
Javed. 2016. Effect of different storage period on egg
opinion on the safety and efficacy of canthaxanthin as a
weight, internal egg quality and hatchability characteristics
feed additive for poultry and for ornamental birds and
of fayumi eggs. Italian J. Anim. Sci. 12:51. https://doi.
ornamental fish. EFSA J. 12:3527. https://doi.org/10.2903/j.
org/10.4081/ijas.2013.e51
efsa.2014.3527
Ko, K. H., I. K. Lee, G. Kim, M. J. Gu, H. Y. Kim, B. Park, T. S.
El-Sabrout, K., S. Aggag, & B. Mishra. 2022. Advanced practical
Park, S. H. Han, & C. H. Yun. 2018. Changes in bursal B
strategies to enhance table egg production. Scientifica 17.
cells in chicken during embryonic development and early
https://doi.org/10.1155/2022/1393392

December 2023 469

 YUSUF ET AL. / Tropical Animal Science Journal 46(4):461-470

life after hatching. Sci Rep. 8:16905. https://doi.org/10.1038/ Saunders-Blades, J. L. & D. R. Korver. 2014. The effect of
s41598-018-34897-4 maternal vitamin D source on broiler hatching egg quality,
Lan, R. X., S. I. Lee, & I. H. Kim. 2017. Effects of Enterococcus hatchability, and progeny bone mineral density and
faecium SLB 120 on growth performance, blood parameters, performance. J. Appl. Poult. Sci. 23:773–783. https://doi.
relative organ weight, breast muscle meat quality, excreta org/10.3382/japr.2014-01006
microbiota shedding, and noxious gas emission in broilers. Schalm, O. W. 1961. Veterinary Hematology. Lea & Febiger,
Poult. Sci. 96:3246-3253. https://doi.org/10.3382/ps/pex101 Philadelphia, US.
Leeson, S. & J. D. Summers. 2005. Commercial Poultry Schisler, D. K. & E. W Kienholz. 1967. Interaction of dietary
Nutrition. 3rd ed. Nottingham Univ Pr., Nottingham (UK). zinc and vitamin D in laying hens. Poult. Sci. 46:918-924.
Li, D., K. Zhang, S. Bai, J. Wang, Q. Zeng, H. Peng, Z. Su, Y. Xuan, https://doi.org/10.3382/ps.0460918
S. Qi, & X. Ding. 2021. Effect of 25-hydroxycholecalciferol Schneider, W. J. 2016. Lipid transport to avian oocytes and to
with different vitamin D3 levels in the hens diet in the the developing embryo. J. Biomed. Res. 30:174-180. https://
rearing period on growth performance, bone quality, doi.org/10.7555/JBR.30.20150048
egg production, and eggshell quality. Agriculture 11:698. Setiyaningsih, N., Sumiati, A. Jayanegara, & W. W. Wardani.
https://doi.org/10.3390/agriculture11080698 2022. Evaluation of Nutricell HyC® (combination of
Lohmann Management Guide. 2019. Lohmann Brown Lite 25OHD3 and vitamin C) supplementation on broiler
Layer Management Guide Cage Housing. Lohmann breeders fertility in laying period. IOP Conference Series:
Tierzucht. Earth and Environmental Science. 1020(1). https://doi.
Martens, P. J., C. Gysemans, A. Verstuyf, & A. C. Mathieu. org/10.1088/1755-1315/1020/1/012016
2020. Vitamin D’s effect on immune function. Nutrients Setiyaningsih, N., Sumiati, A. Jayanegara, & W. W. Wardani.
12:1248. https://doi.org/10.3390/nu12051248 2023. Effects of a vitamins D and C supplement on
Muras, A., M. Romero, C. Mayer, & A. Otero. 2021. performance, hatchability and blood profiles of broiler
Biotechnological applications of Bacillus licheniformis. Crit breeders. J. World Poult. Res. 13:71-80. https://dx.doi.
Rev Biotechnol. 41:609–627. https://doi.org/10.1080/073885 org/10.36380/jwpr.2023.7
51.2021.1873239 Shojadoost, B., S. Behboudi, A. I. Villanueva, J. T. Brisbin, A.
Nabi, F., M. A. Arain, N. Rajput, M. Alagawany, J. Soomro, A. Ashkar, & S. Sharif. 2015. Vitamin D3 modulates the
M. Umer, F. Soomro, Z. Wang, R. Ye, & J. Liu. 2020. function of chicken macrophages. Res. Vet. Sci. 100:45–51.
Health benefits of carotenoids and potential application in https://doi.org/10.1016/j.rvsc.2015.03.009
poultry industry: a review. J. Anim. Physiol. Anim. Nutr. Sinclair-Black, M., R. A. Garcia, & L. E. Ellestad. 2023.
104:1809–1818. https://doi.org/10.1111/jpn.13375 Physiological regulation of calcium and phosphorus
Nascimento, G. R., A. E. Murakami, A. F. Q. M. Guerra, I. C. utilization in laying hens. Front. Physiol. 14:1112499.
Ospinas-Rojas, M. F. Z. Ferreira, & J. C. Fanhani. 2014. https://doi.org/10.3389/fphys.2023.1112499
Effect of different vitamin D sources & calcium levels in USDA. 2000. Egg Grading Manual Handbook. Quorn. Foods
the diet of lay in the second laying cycle. Braz. J. Poult. Sci. Inc., Washington DC (US).
16: 37-42. https://doi.org/10.1590/1516-635x160237-42 Wang, J., L. Qiu, H. Gong, P. Celi, L. Yan, X. Ding, S. Bai, Q.
North, M. O. & D. D. Bell. 1990. Commercial Chicken Zeng, X. Mao, S. Xu, C. Wu, & K. Zhang. 2020. Effect
Production Manual. Springer, New York. of dietary 25-hydroxycholecalciferol supplementation
Obianwuna, U. E., V. U. O. Okoleh, J. Wang, H. Zhang, G. and high stocking density on performance, egg quality,
Qi, K. Qiu, & S. G. Wu. 2022. Natural products of plants and tibia quality in laying hens. Poult. Sci. 99:2608-2615.
and animal origin improve albumen quality of chicken https://doi.org/10.1016/j.psj.2019.12.054
eggs. Frontiers in Nutrition. 9. https://doi.org/10.3389/ Wang, X., P. Zhu, Z. Sun, J. Zhang, & C. Sun. 2021. Uterine
fnut.2022.875270 metabolomic analysis for the regulation of eggshell
Owen, J. A., J. Punt, & S. A Stranford. 2013. KUBY: Immunology. calcification in chickens. Metabolites 11:575. https://doi.
7th Ed. Macmillan Higher Education, Basingstoke (UK). org/10.3390/metabo11090575
Pakpahan, A. M. B., Y. Retnani, R. Mutia, & W. W. Wardani. Wen, J., K. A. Livingston, & M. E Persia. 2019. Effect of high
2023. Evaluation of the physical quality of supplement concentrations of dietary vitamin D3 on pullet and laying
wafer and application of supplement on layer performance. hen performance, skeleton health, eggshell quality, and
Buletin Peternakan 47:190–197. http://dx.doi.org/10.21059/ yolk vitamin D3 content when fed to W36 laying hens from
buletinpeternak.v47i3.84653 day of hatch until 68 wk of age. Poult. Sci. 98:6713–6720.
Pantaya, D. & M. M. D. Utami. 2020. The blood haematological https://doi.org/10.3382/ps/pez386
profile on laying hens that treated by different levels of Yan, L., M. Hu, L. Gu, M. Lei, Z. Chen, H. Zhu, & R. Chen.
yeast supplementation. IOP Conf. Ser.: Earth Environ. Sci. 2022. Effect of heat stress on egg production, steroid
207:012033. https://doi.org/10.1088/1755-1315/207/1/012033 hormone synthesis, and related gene expression in
Plaimast, H., S. Kijparkorn, & P. Ittitanawong. 2015. Effects chicken preovulatory follicular granulosa cells. Animals
of vitamin D3 and calcium on productive performance, (Basel) 12:1467. https://doi.org/10.3390/ani12111467
egg quality and vitamin D3 content in egg of second Yao, L., T. Wang, M. Persia, R. L. Horst, & M. Higgins. 2013.
production cycle fens. Thai J. Vet. Med. 45: 189–195. https:// Effects of vitamin D3-enriched diet on egg yolk vitamin D3
doi.org/10.56808/2985-1130.2635 content and yolk quality. J. Food Sci. 78:178-183. https://
Salles, J., A. Chanet, C. Giraudet, V. Patrac, P. Pierre, M. doi.org/10.1111/1750-3841.12032
Jourdan, Y. C. Luiking, S. Verlaan, C.Migné, Y. Boirie, Zhang, W., K. Y. Zhang, X. M. Ding, S. P. Bai, J. M. Hernande, B.
& S. Walrand. 2013. 1,25(OH)2-vitamin D3 enhances Yao, & Q. Zhu. 2011. Influence of canthaxanthin on broiler
the stimulating effect of leucine and insulin on protein breeder reproduction, chick quality, and performance.
synthesis rate through Akt/PKB and mTOR mediated Poult. Sci. 90:1516–1522. https://doi.org/10.3382/
pathways in murine C 2 C 12 skeletal myotubes. Mol. ps.2010-01126
Nutr. Food Res. 57:2137–2146. https://doi.org/10.1002/ Zhen, W., Y. Shao, X. Gong, Y. Wu, Y. Geng, Z. Wang, & Y. Guo.
mnfr.201300074 2018. Effect of dietary Bacillus coagulans supplementation
Samiullah, S., J. R. Roberts, & K. Chousalkar. 2015. Eggshell on growth performance and immune responses of broiler
color in brown-egg laying hens - a review. Poult. Sci. chickens challenged by Salmonella enteritidis. Poult. Sci. 97:
94:2566-2575. https://doi.org/10.3382/ps/pev202 2654–2666. https://doi.org/10.3382/ps/pey119

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