Effects of Calcium Carbonate Particle Size, Phytase and Midnight Feeding on Performance, Egg and Bone Quality and Blood Parameters in Laying Hens

ABSTRACT

Background

To achieve maximum production in laying hens, calcium (Ca) and phosphorus (P) are two important minerals in the diet. Nutritional solutions such as adding coarser Ca particles in the afternoon or considering the light hours during the night to feed the birds might improve the eggshell quality and reduce Ca mobilization from the bone.

Objectives

This experiment aimed to study the effect of calcium carbonate particle size (CCps), phytase (Pht) and midnight feeding (MF) on the performance, egg and bone quality and blood parameters in laying hens from 60 to 70 weeks of age.

Methods

A total of 720 Hy‐line (W‐36) laying hens were arranged in a factorial arrangement of 2 × 2 × 2 based on a completely randomized design with two types of CCps (100% fine and 75% coarse + 25% fine), two levels of Pht (0 and 300 FTU/kg) and two states of feeding (MF and without MF). The experimental period lasted 12 weeks from 60 to 70 weeks of age.

Results

During the trial, the significant impact of CCps on the feed conversion ratio (FCR) and breaking strength (BS) of the tibia was obtained (p < 0.05). FCR, eggshell percentage and the percentage of tibia ash, Ca and P were affected by Pht supplementation (p < 0.05). MF affected egg weight (EW), eggshell percentage and serum concentration of Ca, P and alkaline phosphatase (ALP) (p < 0.05). The interaction effects of CCps and Pht were significant for Ca level in serum (p < 0.05).

Conclusions

In general, the use of Pht in the diets improved the FCR and quality of eggshell and tibia, and diets containing Pht and coarser CCps increased blood Ca levels. Despite the improvement of egg quality by MF, serum parameters showed a significant decrease.

• 720 Hy‐line (W‐36) laying hens were assigned to eight treatments, with two levels calcium carbonate particle size (fine and coarse), phytase (0 and 300 FTU/kg diet) and midnight feeding (with and without) from 60 to 72 weeks of age.

• The use of coarser calcium carbonate particles, phytase and midnight feeding improved the performance, eggshell percentage, tibia characteristics and serum parameters.

• Midnight feeding caused a significant decrease in blood calcium and phosphorus levels.

1. Introduction

One of the important nutritional issues related to laying hens is their high calcium (Ca) requirements because the presence of this mineral during the night is necessary for the production of eggshells (Sarmiento‐Garcia et al. 2022). Poultry feed can meet around 60% of Ca required by birds (Nys and Guoyt 2011). However, due to the asynchronism between dietary Ca availability during the day and the high Ca requirements for eggshell production at night, surplus Ca is mobilized from the medullary bone (Hervo et al. 2023). To supply the required Ca and to decrease the split between the supply and demand for this mineral, coarse calcium carbonate (CC) is usually added to the poultry diet (Molnar et al. 2018). It has been stated that the retention time of CC in the gizzard is longer than that of fine particles. Due to the acidic environment of the gizzard and the longer retention time of CC in it, the dissolution of Ca is slower, and the release of Ca in the duodenum is prolonged during the night, and as a result, the mobilization of Ca from the bone is reduced (Fleming 2008). On top of that, a few studies have shown that night access to Ca plays an important role in improving eggshell quality (Farmer et al. 1986). It has been reported that nutritional strategies such as adding coarser Ca particles in the afternoon or considering two hours of lightness during the night (birds are fed at the same time) improved the eggshell quality and reduced the Ca mobilization from the bones and plasma phosphorus (P). As a result, the excretion of excess P will decrease (Aldridge et al. 2022; Alfonso‐Carrillo et al. 2021).

On the other hand, about 60%–70% of P in cereal grains forms a phytic acid complex due to binding to myo‐inositol, which greatly decreases its bioavailability for animals (Abbasi et al. 2019). Therefore, poultry farmers are eager to use phytase (Pht) because it increases the bioavailability of P from phytic acid and diminishes the excretion of P (Gupta et al. 2015). It has been reported that Pht improved the nutritional value of feed and reduced the pollution caused by excessive accumulation of P in soil and water (Nahm 2002). Due to the importance of Ca and P in the laying hens' diet and their prominent effects on the eggshell quality and bird performance, as well as due to the effects of midnight feeding (MF) on the productivity of these two important minerals, the purpose of this study was to simultaneously investigate the effects of Pht, CC particle size (CCps) and MF on the performance of laying hens.

2. Materials and Methods

2.1. Birds Housing and Management

In this regard, 720 leghorn laying hens (Hy‐lineW36) at 60‐week‐old were randomly allocated in three‐storey cages and subjected to eight experimental treatments with six repetitions (15 hens per replication), using a completely randomized design (CRD). The hens were kept in wire cages 52‐cm long, 34‐cm wide and 30‐cm high on three floors. The experiment had a 3‐week adaptation phase of the bird to the diet and house conditions. Temperature was maintained at approximately 20°C with 15 h of constant light, 3.5 h of darkness, 2 h of midnight light and 3.5 h of darkness per day. Birds were checked and eggs were collected and recorded daily. Feed and water were provided ad libitum throughout the experiment. The experimental period lasted 12 weeks from 60 to 70 weeks of age.

2.2. Treatments and Diets

Experimental diets were adjusted based on the requirements recommended for the strain ‐Hyline International 2020‐by using UFFDA software, the components of which are presented in Table 1. A 2 × 2 × 2 factorial arrangement was applied with two types of CCps (100% fine [< 0.5 mm] and 75% coarse [2–4 mm] + 0.25% fine), two levels of Pht (0 and 300 FTU/kg; Ronozyme HiPhos, 6‐phytase produced by the strain of Aspergillus oryzae) and two states of feeding (with and without MF). Treatments included: (1) fine—no Pht—MF, (2) fine—no Pht—without MF, (3) fine—Pht—MF, (4) fine—Pht—without MF, (5) coarse—no Pht—MF, (6) coarse—no Pht—without MF, (7) coarse—Pht—MF, (8) coarse—Pht—without MF. In the groups receiving midnight diets, in the morning, Ca concentration was decreased, and available P (aP) was increased, while in the evening, Ca concentration was enhanced and aP was reduced by 20%.

TABLE 1.

Ingredients and chemical composition of the experimental diets (as‐fed basis).

Ingredients (g/kg)	Control diet	Morning diet	Afternoon diet
Corn	613.80	618.30	618.30
Soybean meal (44% CP)	238.60	238.60	238.60
Soybean oil	19.80	23.00	23.00
Limestone	100.80	76.30	125.40
Dicalcium phosphate	12.90	16.20	9.50
Common salt	3.50	3.40	3.50
Vitamin premix a	2.50	2.50	2.50
Mineral premix	2.50	2.50	2.50
dl‐Methionine	1.10	1.10	1.10
Nutrient composition (%)
Metabolizable energy (kcal/kg)	2800	2800	2800
Crude protein	15.20	15.20	15.20
Calcium	4.18	3.34	5.02
Non‐phytate phosphorus	0.36	0.43	0.29
Sodium	0.16	0.16	0.16
Lysine	0.70	0.70	0.70
Methionine	0.35	0.35	0.35
Methionine + cystine	0.57	0.57	0.57
Threonine	0.49	0.59	0.39

Note: Limestone particle size: (100% fine [< 0.5 mm] and 75% coarse [2–4 mm] + 0.25% fine); two levels of phytase (0 and 300 FTU/kg [Ronozyme HiPhos, 6‐phytase produced by the strain of Aspergillus oryzae]). In the groups receiving midnight diets, in the morning, Ca concentration decreased and available P (aP) increased, while in the evening, Ca concentration enhanced and aP reduced by 20%.

^a Layer premix contains the following, expressed per kg: vitamin A, 25,000,000 UI; vitamin D3, 860,000 UI; vitamin E, 20,000 UI; vitamin K3, 1000 mg; pantothenic acid, 5000 mg; pyridoxine, 1600 mg; thiamine, 1000 mg; vitamin B12, 5.2 mg; vitamin PP, 20,000 mg; biotin, 60 mg; folic acid, 600 mg; Fe, 10,000 mg; Zn, 14,000 mg; Mn, 16,000 mg; Cu, 3200 mg; I, 400 mg; Se, 40 mg.

2.3. Performance

Performance traits including egg production (EP; %), egg weight (EW; g), egg mass (EM; g/day), daily feed consumption (DFC; g/day) and feed conversion ratio (FCR; g/g) were investigated. Total produced eggs were collected and weighed; their number was recorded daily and at a certain hour (at 2:00 pm) and the percentage of EP was determined based on the hen day. To calculate the EW, all the eggs produced in each experimental unit were weighed every day, and then by dividing the weight by the number of eggs, the average EW of each experimental unit was calculated weekly. EM was obtained from the product of average EW and EP percentage. The feed consumption of each experimental unit was measured from the difference between the amount of the distributed diet and the amount of remaining diet. FCR was determined by dividing the feed consumption by the EM (Khoshbin et al. 2023).

2.4. External and Internal Attributes of Egg Quality

To check the characteristics of eggs, at the end of each week, two eggs from each replicate were randomly selected (n = 96). The length and width of the egg were measured using a digital caliper (Ogawa, Japan) with an accuracy of 0.01 mm, and then the egg shape index was obtained by dividing the width of the egg by its length. To measure the eggshell percentage, after breaking the eggs, the shell was separated and kept in the oven for 72 h at 65°C, and after taking it out of the oven and cooling down, their weight was measured, and it was expressed as a percentage of EW. The index of albumen and yolk was calculated using the following formulas:

$$\begin{array}{ccc}& & \mathrm{Albumen}\mathrm{index}\left(\%\right)\hfill \\ & & =\frac{\mathrm{Albumen}\mathrm{height}\left(\mathrm{mm}\right)}{\mathrm{Albumen}\mathrm{length}\left(\mathrm{mm}\right)+\mathrm{Albumen}\mathrm{width}\left(\mathrm{mm}\right)/2}\times 100\hfill \end{array}$$

$$\mathrm{Yolk}\mathrm{index}\left(\%\right)=\frac{\mathrm{Yolk}\mathrm{height}\left(\mathrm{mm}\right)}{\mathrm{Yolk}\mathrm{diameter}\left(\mathrm{mm}\right)}\times 100$$

2.5. Bone Parameters

At 72 weeks of age, six birds from each treatment (one bird from each cage) were randomly selected and slaughtered (n = 48). The left tibia from each bird was removed to evaluate the percentage of ash, Ca, P and breaking strength (BS) at 72 weeks of age. After separating the meat and soft tissues from each tibia, they were dried in the oven and the length and diameter of them in the diaphysis area were measured using a digital micrometre with an accuracy of 1 micron (series 500, Mitutoyo, Tokyo, Japan). Bones were weighed, and an Instron Universal Testing Machine (Model H5KS, Tinius Olsen Company) determined BS. The percentage of ash, Ca and P of the tibia was determined according to the method of Noruzi et al. (2023).

2.6. Blood Parameters

Blood samples were collected from the wing vein of six hens per treatment of 72 weeks old (n = 48). For haematological assays, 2 mL of sample was poured into an anticoagulant (EDTA Sigma‐Aldrich, E6758) containing tube and transferred to the laboratory. Blood samples were centrifuged at 1500 × g for 15 min to separate the serum, which was kept at room temperature until analysis. Serum total protein, albumin, Ca, P, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were determined using appropriate kits (Pars Azmoon Kit, Tehran, Iran).

2.7. Statistical Analysis

This experiment was performed based on a CRD in a factorial arrangement of 2 × 2 × 2 (Equation 1) with six replicates by the GLM procedure of SAS 9.4 (2012) software. Outcomes are indicated as means ± standard error of the mean (SEM). All data normalization was assessed by the Shapiro–Wilk test. The significance of treatment differences was analysed using ANOVA. The means were compared by Tukey's test (p < 0.05).

$$X_{ijkl}=\mu +A_j+B_k+C_l+A{B}_{jk}+A{C}_{jl}+B{C}_{kl}+AB{C}_{jkl}+{\epsilon }_{ijkl}$$ (1)

where X_ijkl is an observation, μ is the experimental mean, A is CCps effect (coarse and fine), B is Pht effect (0 and 300 FTU/kg), C is MF effect (with or without MF), AB is the two‐way interaction effects of CCps and Pht, AC is the two‐way interaction effects of CCps and MF, BC is the two‐way interaction effects of Pht and MF, ABC is the three‐way interaction effects of CCps, Pht and MF and $\epsilon$ is the experimental error.

3. Results

3.1. Performance

The data related to the main and interaction effects of CCps, Pht and MF on the EP, EW, EM, DFC and FCR are indicated in Table 2. The outcomes illustrated that increasing the size of CC particles reduced significantly FCR from 2.01 to 1.95 (p < 0.05). Regarding the main effect of Pht, the lowest FCR (1.94) was observed in the hens receiving diets containing enzyme (p < 0.05). Concerning the main effect of MF, EW rose considerably in birds that feed during the night (p < 0.05).

TABLE 2.

Main and interaction effects of carbonate calcium particle size, phytase and midnight feeding on the performance traits from 60 to 72 weeks of age.

Main effects	EP (%)	EW (g)	EM (g/day)	DFC (g/day)	FCR (g/g)
CCps
Fine	76.63	63.88	50.17	99.93	2.01a
Coarse	75.86	64.29	49.47	99.42	1.95b
p value	0.62	0.20	0.60	0.60	0.04
SEM	1.104	0.223	0.932	0.639	0.017
Pht (FTU/kg diet)
0	76.51	64.31	49.82	99.87	2.00a
300	75.98	63.86	49.83	99.48	1.94b
p value	0.70	0.17	0.99	0.70	0.03
SEM	1.104	0.223	0.932	0.639	0.018
MF
No	75.71	63.51b	49.46	99.47	1.99
Yes	76.78	64.67a	50.18	99.88	1.97
p value	0.50	0.007	0.60	0.70	0.20
SEM	1.104	0.223	0.931	0.639	0.017
			p value
Interactions
CCps*Pht	0.90	0.07	0.50	0.10	0.40
CCps*MF	0.60	0.60	0.81	0.80	0.69
Pht*MF	0.94	0.80	0.90	0.40	0.64
CCps*Pht*MF	0.57	0.85	0.73	0.90	0.69

Note: Means within a column without a common superscript alphabet significantly differ (p < 0.05).

Abbreviations: CCps, calcium carbonate particle size; DFC, daily feed consumption; EM, egg mass; EP, egg production; EW, egg weight; FCR, feed conversion ratio; MF, midnight feeding; Pht, phytase enzyme (Ronozyme HiPhos, 6‐phytase produced by the strain of Aspergillus oryzae); SEM, standard error of the mean.

3.2. External and Internal Attributes of Egg Quality

The effects of CCps, Pht and MF on the yolk index, albumen index, shape index and eggshell percentage are shown in Table 3. According to this table, eggshell percentage enhanced remarkably in birds that received diets containing Pht or had the MF program (p < 0.05).

TABLE 3.

Main and interaction effects of carbonate calcium particle size, phytase and midnight feeding on the egg quality from 60 to 72 weeks of age.

Main effects	Yolk index	Albumen index	Shape index	Eggshell percentage
CCps
Fine	38.52	10.05	75.43	8.61
Coarse	38.59	10.08	75.65	8.54
p value	0.89	0.78	0.55	0.50
SEM	0.318	0.176	0.256	0.072
Pht (FTU/kg diet)
0	38.47	10.10	75.34	8.45b
300	38.64	10.09	75.74	8.71a
p value	0.70	0.78	0.28	0.02
SEM	0.318	0.176	0.254	0.072
MF
No	38.46	9.91	75.32	8.46b
Yes	38.65	10.17	75.75	8.69a
p value	0.65	0.30	0.25	0.03
SEM	0.318	0.176	0.256	0.072
	p value
Interactions
CCps*Pht	0.20	0.15	0.79	0.95
CCps*MF	0.19	0.69	0.19	0.69
Pht*MF	0.55	0.87	0.06	0.83
CCps*Pht*MF	0.65	0.37	0.60	0.37

Note: Two eggs were selected from each cage (96 eggs in total). Means within a column without a common superscript alphabet significantly differ (p < 0.05).

Abbreviations: CCps, calcuim carbonate particle size; MF, midnight feeding; Pht, phytase enzyme (Ronozyme HiPhos, 6‐phytase produced by the strain of Aspergillus oryzae); SEM, standard error of the mean.

^a,b

3.3. Bone Parameters

The results of bone characteristics at 72 weeks of age are displayed in Table 4. The table showed that BS of tibia was affected by the main effect of CCps, so that the tibia of hens consuming coarse particles showed greater BS (p < 0.05). With respect to the main effects of Pht, tibia ash, Ca and P percentages, they increased by using Pht (p < 0.05).

TABLE 4.

Main and interaction effects of carbonate calcium particle size, phytase and midnight feeding on the tibia characteristics from 60 to 72 weeks of age.

Main effects	Length (mm)	Diameter (mm)	Breaking strength (N)	Ash (%)	Calcium (%)	Phosphorus (%)
CCps
Fine	115.02	6.54	182.78b	51.54	21.81	9.30
Coarse	115.68	6.65	192.15a	52.13	22.44	9.58
p value	0.45	0.56	0.03	0.25	0.12	0.07
SEM	0.589	0.129	2.794	0.355	0.274	0.111
Pht (FTU/kg diet)
0	115.87	6.57	184.49	51.10b	21.66b	9.28b
300	114.83	6.62	190.44	52.57a	22.57a	9.60a
p value	0.23	0.80	0.15	0.006	0.03	0.04
SEM	0.589	0.129	2.863	0.355	0.274	0.111
MF
No	115.27	6.46	183.61	51.23	21.55	9.31
Yes	115.43	6.73	191.32	51.44	21.70	9.57
p value	0.85	0.16	0.06	0.090	0.11	0.10
SEM	0.574	0.129	2.794	0.355	0.274	0.111
			p value
Interactions
CCps*Pht	0.72	0.50	0.17	0.67	0.58	0.70
CCps*MF	0.33	0.18	0.12	0.54	0.54	0.33
Pht*MF	0.87	0.10	0.64	0.21	0.91	0.30
CCps*Pht*MF	0.84	0.72	0.89	0.47	0.43	0.27

Note: Total number of samples = 48. Means within a column without a common superscript alphabet significantly differ (p < 0.05).

Abbreviations: CCps: calcuim carbonate particle size; MF: midnight feeding; Pht: phytase enzyme (Ronozyme HiPhos, 6‐phytase produced by the strain of Aspergillus oryzae); SEM: Standard error of the mean.

3.4. Blood Parameters

The effects of treatments on blood factors are illustrated in Table 5. MF increased substantially ALP and reduced Ca and P levels compared to hens that did not receive MF (p < 0.05). In relation to two‐way interactions, treatments containing fine particle size with or without Pht had the lowest level of Ca (16.42 and 15.96 mg/dL, respectively), while groups containing Pht with coarse particle size of CC showed the highest concentration of Ca in serum (17.28 mg/dL) (p < 0.05) (Figure 1).

TABLE 5.

Main and interaction effects of carbonate calcium particle size, phytase and midnight feeding on the blood parameters from 60 to 72 weeks of age.

Main effects	Calcium (mg/dL)	Phosphorus (mg/dL)	Total protein (g/dL)	Albumin (g/dL)	AST (U/L)	ALT (U/L)	ALP (U/L)
CCps
Fine	16.23	5.70	6.65	2.24	212.48	8.70	572.87
Coarse	17.75	6.35	7.08	2.23	221.69	9.38	612.91
p value	0.03	0.06	0.09	0.95	0.40	0.20	0.18
SEM	0.233	0.163	0.124	0.083	7.580	0.368	20.467
Pht (FTU/kg diet)
0	16.59	5.92	6.73	2.27	207.58	8.89	561.26
300	17.37	6.30	7.00	2.20	226.59	9.19	604.52
p value	0.02	0.12	0.12	0.58	0.09	0.57	0.10
SEM	0.233	0.163	0.124	0.083	7.580	0.359	20.467
MF
No	17.46a	6.40a	6.78	2.15	214.08	8.75	556.28b
Yes	16.50b	5.82b	6.95	2.32	220.08	9.33	629.49a
p value	0.005	0.02	0.34	0.17	0.59	0.27	0.01
SEM	0.233	0.163	0.124	0.083	7.397	0.368	20.467
				p value
Interactions
CCps*Pht	0.04	0.61	0.08	0.37	0.85	0.57	0.62
CCps*MF	0.20	0.25	0.18	0.08	0.82	0.27	0.67
Pht*MF	0.26	0.66	0.72	0.07	0.71	0.13	0.43
CCps*Pht*MF	0.75	0.39	0.17	0.30	0.50	0.49	0.24

Note: Total number of samples = 48. Means within a column without a common superscript alphabet significantly differ (p < 0.05).

Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CCps, calcium carbonate particle size; MF, midnight feeding; Pht, phytase enzyme (Ronozyme HiPhos, 6‐phytase produced by the strain of Aspergillus oryzae); SEM, standard error of the mean.

FIGURE 1.

The two‐way interaction effects of calcium carbonate particle size (fine or coarse) and phytase (Ronozyme HiPhos, 6‐phytase produced by the strain of Aspergillus oryzae: 0 and 300 FTU/kg diet) on the serum calcium level in 72‐week‐old laying hens.

4. Discussion

In general, there is no complete and clear information about the effects of CCps along with Pht and MF on the performance of laying hens. However, the current study showed the positive effects of coarse particles and Pht (300 FTU/kg) on the FCR. Data showed that increasing the size of CC particles improved significantly hens' FCR (2.01 in fine particles compared to 1.95 for coarse particles). Moreover, birds consuming diets containing Pht showed better FCR compared to those that did not utilize Pht (1.94 vs. 2.00, respectively). In accordance with these results, Hervo et al. (2023) reported that with the increase in the CCps (2–4 mm), the FCR improved significantly in 37‐week‐old hens (from 1.62 to 1.57). Skrivan et al. (2010) showed that enhancing the CCps from 0.8 to 2 mm increased EP and reduced the FCR in younger and older hens compared to the fine particle size (< 0.5 mm). In contrast, other studies did not observe any significant effect of CCps on the hens' performance (Oliveira et al. 2013; Saunders‐Blades et al. 2009). They stated that changing in the CCps did not considerably affect feed consumption and consequently hens' performance. Because even birds exposed to different CCps received enough nutrients for their efficient performance. It has been reported that the source of Ca and its solubility in the gizzard affect the quality of eggshell. The slow dissolution of Ca sources (e.g., limestone) increased the availability of Ca during eggshell calcification and thus decreased the bone Ca and P mobilization (Skrivan et al. 2010). It has been stated that limestone with larger particle size and lower solubility (in vitro condition) increased Ca retention due to longer retention time in the gizzard (Anwar et al. 2017). In agreement with this study, Habibollahi et al. (2019) reported that the use of Pht (250 FTU/kg) in the layers diet caused a significant improvement in the FCR. Several factors play a role in the effect of Pht on the birds' performance, which can be mentioned as the Ca to P ratio, the amount of vitamin D, organic acids, the interference of metal ions, and feeding and light programs (Selle and Ravindran 2007). Cabuk et al. (2004) observed that the use of Pht (300 FTU/kg) in the diet (54‐week‐old Nick‐Brown) improved the hens' FCR. Bello et al. (2020) also reported the positive effects of Pht on the birds FCR. They stated that supplementing diets with Pht reduced the adverse effects of dietary Ca and P deficiency on body weight. Moreover, it has been said that inclusion of Pht increased mineral availabilities in the digestive tract of laying hens (Bello et al. 2020). It has been stated that the positive effects of Pht on the disappearance of phytic P were more significant when diets with coarse and fine limestone were used compared to only fine particles (Jafari Arvari et al. 2023). Although this parameter was not measured in the present experiment, other studies have shown that the formation of the Ca–phytate complex was largely influenced by the size of CC particles in the broilers, and CC with smaller particles facilitated the formation of this complex (Manangi and Coon 2007).

Eggshell weight increased significantly in birds receiving MF (Van Emous and Mens 2021). It has been said that eggshell quality could be improved by turning the lights on at midnight and operating the feeder for 45 min (Harms et al. 1996). In the current study, hens that consumed a night diet had higher EW compared to those that did not have night feed (64.76 vs. 63.51 g, in turn). During the formation of the eggshell, necessary Ca was mobilized from the bones (Zhang et al. 2024). Within this process, P was also released, which was excreted through urine (Nys and Guoyt 2011). As a result, P requirement increased during eggshell formation. In the present trial, Pht improved eggshell percentage, so that eggshell percentage was 8.71% and 8.45% for diets containing Pht and diets without Pht, respectively. Various results have been published regarding the effects of Pht on the eggshell quality. Some studies have reported advantageous impacts (Habibollahi et al. 2019) and some ineffectiveness of Pht (Pirzado et al. 2024; Waters et al. 2024). In addition, the MF program significantly enhanced the eggshell percentage (8.69% in birds that had midnight feed and 8.46% for those that did not receive midnight diets). It has been reported that plasma P levels were higher when eggs were laid in the morning, and eggshell percentage is higher in the morning eggs (from night‐fed hens) (Molnar et al. 2018). Lee and Ohh (2002) stated that a decrease in Ca level in the morning and an increase in its level in the afternoon led to a higher shell thickness and BS compared to fixed Ca levels.

In the present study, BS of tibia was considerably affected by enhancing the CCps. In accordance with our results, Oliveira et al. (2013) reported similar outcomes. It can be stated that the addition of a Ca source with larger particles may help maintain bone integrity in laying hens (Oliveira et al. 2013). Saunders‐Blades et al. (2009) reported that diets with larger CCps decreased osteoporosis and improved poultry welfare at the end of the laying cycle. However, Jardim Filho et al. (2005) did not observe any significant effect of CCps. They attributed this diversity to factors such as the birds' age and the equipment used to measure tibia strength (Jardim Filho et al. 2005). According to the results of the present study and other studies, since there are diverse reports of increased bone resistance at the end of the production cycle in poultry fed with large CCps, it can be concluded that maintaining bone quality in poultry is an advantage, which is obtained by changing the particle size of the CC in their diet (Liu et al. 2023). The Pht supplementation improved the percentages of ash, Ca and P in bone. Zhai et al. (2022) reported that the use of Pht significantly increased the percentage and weight of bone Ca and P, while bone ash was significantly improved only by the control diets (without Pht). It has been found that increasing the level of dietary P due to the use of Pht significantly enhanced the concentration of P in the tibia and plasma (El Boushy 1979). Due to the relationship between dietary P, plasma P and tibia P in laying hens, it can be said that the P released by Pht reduced the Ca to P ratio in the plasma towards the optimal ratio for more bone mineralization (Boorman and Gunaratne 2001). Mansoori and Modirsanei (2015) also reported that Pht increased the tibia relative weight, ash content and BS. Lei et al. (2011) evaluated the effects of complete removal of P from the diet and reduction of energy and protein without and with Pht supplementation on performance, egg quality and bone composition of 56‐week‐old laying hens. They indicated that feed consumption, EP, body weight and tibia ash percentage reduced by P removal but restored by Pht inclusion. They declared that eliminating P from the diet diminished the quality of the tibia, while adding Pht improved the tibia integrity (Lei et al. 2011). Other studies (Hughes et al. 2008; Junqueira et al. 2010; Panda et al. 2005) also reported the positive effects of Pht on the tibia and its ash content.

To evaluate the metabolism of birds, an analysis of the biochemical composition of the blood is performed (So et al. 2009). It has been said that larger particles pass through the intestine slowly due to low solubility; as a result, Ca is available to the birds for a longer period of time (Tunc and Cufadar 2014). In line with present results, Akpiruo (2020) said that birds fed diets containing large and medium eggshell as a source of Ca had the highest serum Ca. It has been declared that in older birds, Ca metabolism takes longer. Therefore, limestone with coarse particles is preferred, as it provides a continuous supply of Ca (Pelicia et al. 2011). In the present study, the Ca level in serum increased significantly due to the use of Pht. This increment could possibly be due to the hydrolysis of phytate by Pht in the diets, which helps in the digestion and absorption of Ca and P (Lim et al. 2020). These results were confirmed by the data obtained by Hassanien and Elnagar (2011), which stated that the serum Ca concentration raised with Pht supplementation and showed that Pht improved Ca bioavailability in laying hens. In addition to the main effects of CCps and Pht, their interaction was significant as well. Hens that consumed diets with CC coarse particles and Pht had the highest Ca levels (17.28 mg/dL) in the serum samples. Indeed, the effect of Pht on phytate and the release of Ca in the blood (Lim et al. 2020), and on the other hand, the continuous supply of Ca through the consumption of large limestone particles (Pelicia et al. 2011), probably increased the serum Ca level. In the current study, MF enhanced ALP levels and reduced Ca and P concentrations in the blood samples. The level of ALP is considered an indicator for investigating hyperparathyroidism in humans and abnormal bone metabolism in laying hens (Iwanaga et al. 2016; Wei et al. 2021). The increase in ALP activity in hens that had MF can be related to the increase in Ca mobilization from bones (Wei et al. 2021). Therefore, the relatively higher plasma ALP activity and decreased plasma Ca and P levels in night‐fed hens may be a result of increased Ca deposition in the eggshell (Cransberg et al. 2001). In accordance with our outcomes, Attia et al. (2010) reported that serum concentrations of total protein, AST and ALT were not affected by Pht supplementation in the diet.

5. Conclusion

Investigating the main and interaction effects of the factors (CCps, Pht and MF) showed an increase in serum calcium levels because of the consumption of diets containing Pht and coarse particles of CC. However, to improve other measured traits (performance, egg quality and tibia characteristics), assessing the effects of one of the factors could be sufficient. Finally, to ameliorate the performance and other parameters in the study, the authors recommended the use of each of the independent variables alone, except for the increase in serum calcium level. In addition to the factors measured in the present experiment, factors such as the age of the hens, their physiological conditions, diet composition and the level of Pht should be considered.

Author Contributions

Vahid Salehi: data curation, funding acquisition. Reza Vakili: conceptualization, investigation, writing–original draft, methodology, validation, visualization, writing–review and editing, project administration, resources, supervision. Mahdi Elahi Torshizi: software, formal analysis.

Ethics Statement

We hereby declare all ethical standards have been respected in preparation of the submitted article.

Conflicts of Interest

The authors declare no conflicts of interest.

Peer Review

The peer review history for this article is available at https://publons.com/publon/10.1002/vms3.70248.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.