Introduction

Poultry diets include phosphorus (P), which is the third most costly component behind protein and energy; therefore, it was necessary to use alternatives that improve the availability of phosphorus (1), such as mineral nanoparticles that improved the intestine’s degree of availability and absorption. Also, it helps to decrease the supplementation cost and the environmental pollution (2).  Minerals in chicken feed mostly come from dicalcium phosphate (DCP), which is essentially for metabolism and development (3-4). Nano minerals have a greater utilization rate compared to conventional inorganic and organic minerals (5- 6).).  Nano minerals have unique properties compared to macromolecules (7-10). The size of the nanominerals' particles ranges from one to one hundred nanometers (11). Due to the close connection between phosphorus (P) and calcium (Ca) minerals, imbalances in one mineral can impact the metabolism of the other (12). Factors affecting the digestibility of phosphorus in the feed include the calcium source's particle size, the limestone's solubility, and the overall calcium content in the feed (13). Recently, several studies have focused on using nano calcium phosphate in poultry. Broiler chickens' immunological responses and functional intestinal morphology might be enhanced by applying 40% and 60% amounts of nano-dicalcium phosphate, with no detrimental effects on hematological parameters (14). For hence, the optimal dosage for the broiler chicks' digestion, absorption, and breast phosphorus content was 0.35% nano calcium phosphate (3). Furthermore, when broiler diets contain nano dicalcium phosphate, it is possible to successfully reduce the dietary dicalcium phosphate by 75% without adversely affecting broiler performance, while also reducing excreted calcium and phosphorus by 50%, thereby decreasing environmental pollution caused by poultry (15). Similarly, when used as nanoparticles, the dietary dicalcium phosphate level was successfully reduced from 1.75 to 0.44%, improving the characteristics of tibia bone (16). In addition, a small amount of NHA could be added to broiler diets as a substitute source of calcium and phosphorus (17). Additionally, calcium-phosphorus compounds have shown no adverse effects on the health of birds, improved bone quality and production performance, allowed for the use of lower dosages of nanosources, and reduced the amount of calcium and phosphorus in excreta (by around 50%) (18). The Haugh unit (HU) values of Japanese quail eggs increased (P≤0.05) by 50% NCaP in the diet (19). Supplementing laying hens' diets with nano minerals improved the quality of their eggs (20). Omara et al. (21) demonstrated that the dietary supplementation of broilers with nano phosphorous did not affect the abundance of D-24-hydroxylase mRNA. The Ca and P serum concentrations were influenced by dietary NCaP (22).

These findings indicated that NCaP is a promising alternative mineral source for poultry diets. Therefore, the purpose of this research was to assess the nano source compared to the traditional source of DCP on Lohmann Brown hen's performance, egg quality, tibia bone characteristics, and gene expression for the kidney's and small intestine's P and Ca co-transporters.

Materials and methods

Ethical approval

The Institutional Animal Care and Use Committee (IACUC) of Cairo University granted ethical permission for this work (CU/II/F/7/21).

Materials

The dicalcium phosphate (DCP) was purchased from chemical company in Egypt, while Calcium phosphate nanopowder (NCaP)-hydroxyapatite (Ca₁₀(PO4)₆(OH)₂) product  (American Elements Co., Los Angeles, CA) contained 98.5% calcium phosphate (39.90% Ca and 18.50% P) with a Ca:P ratio of 2.15:1.00. The particle size ranged from 500 to 1,000 μm for Dical P and <100 nm for NCaP.

Birds, managements and diets

The experiment was place at Cairo University's Poultry Farm, which is a component of the Animal Production Department inside the Faculty of Agriculture., Giza, Egypt and lab analysis were conducted in labs of Cairo University and National Research Centre, Egypt. There were five groups of 27-week-old five hundred Lohmann Brown laying hens with an initial body weight of about 1854 g (control diet + 4 NCaP diets). Each of the one hundred birds/treatments was distributed into 10 pens (10 hens/pen).  Birds fed a basal diet based on corn-soybean meal covered all nutrients according to the strain guide except DCP, which was added in 100% (T1, Control) and different levels of 100, 75, 50, and 25% (14, 29, 35) of NCaP in T2, T3, T4, and T5, respectively (Table 1). Hens were exposed to 16 h light and 8 h dark, with temperature maintained at 20–24°C, relative humidity 55–65%, and a tunnel ventilation system was in place to ensure adequate air exchange and air quality within the hall to keep ammonia levels below 10 ppm. The experiment lasted for 16 weeks at 43 weeks of age.

Hen’s productive performance

Egg number (EN) was daily recorded to calculate hen-day egg production (EP) for each replicate, where EP % = EN/daily layers number X100. Average egg weight (EW) was recorded every period (4 weeks) in order to determine the egg mass (EM), where EM (g/hen/day) = {(EP X EW)/100}. Average daily feed intake (FI) was calculated every period (g/hen/day), Feed intake (FI)=feed offered− remaining feed . Feed conversion ratio (FCR) = FI (g.feed) / EM (g.egg). All egg production performance parameters were calculated according to the standard procedures outlined in the Lohmann Brown management guide (23).

Table 1: Ingredients and nutrients composition of experimental diets

* Vitamin and mineral premix at 0.3% of the diet supplies the following per Kg of the diet: Vitamin A 10000 I.U, Vitamin D₃ 3000 I.U, Vitamin E 20mg, Vitamin K₃ 3mg, Vitamin B₁ 2mg, Vitamin B₂ 6mg, Vitamin B₆ 5mg, Vitamin B₁₂ 20mg,  Pantothenic acid 10mg, Folic acid 1mg, Biotin 5mg, niacin 66mg, Manganese 100mg, Iron 100mg, Zinc 75mg, Copper 8mg, Iodine 45mg, Selenium 10mg, Cobalt 10mg.

Quality of eggs

We sampled 25 eggs from each treatment at the conclusion of the trial to find out how good the eggs were.  The weight of the egg shell was measured in grams after the inside membrane was washed and dried overnight at temperatures ranging from 60 to 70°C. The thickness of each eggshell was measured in micrometers using a digital micrometer vernier caliper on four separate areas: the blunt end, the sharp end, the two sides of the egg's equator, and the egg itself. The egg shape index (SI) was derived by dividing the egg length by width, the percentage shell was computed using the micrometer-measured height of albumen, and the Haugh units were determined according to the method of Eisen et al. (24). The color of the egg yolk was found by comparing it to 15 different bands of the color spectrum and by utilizing the Roche enhanced yolk color fan.  According to AOAC (25) total lipid (g/100g) and total cholesterol (mg/Kg) were measured in of egg yolk.

Tissue sampling

Upon completion of the experiment (43 weeks of age), 20 birds from each treatment were slaughtered, and the right tibia bone was removed for analysis of several features. Additionally, a small portion of kidney and the intestinal duodenum and jejunum had been taken to analyze gene expression.

Blood biochemistry

In order to get serum, blood samples were centrifuged at 2000 rpm after being drawn from the wing vein in plain tubes at the end of the experiment. Samples serum were used to determine the Ca, P, sodium, potassium, chloride, alkaline phosphatase, aspartate aminotransferase, glucose, cholesterol, total protein, albumin and  uric acid using commercial diagnostic kites (Biomed diagnostics, Germany) by spectrophotometer (JEN WAY 3600), while parathyroid hormone (PTH) levels were determined by ELISA technique using (MyBioSource chicken PTH, E-EL-H0092) ELISA kit .      

Tibia bone characteristics

The right tibia has been taken out and ready for the various measurements. Diethyl ether was used to remove the adherent flesh from the tibia bone. The tibia bones' weight was determined, and they were oven-dried for 3 hours at 105°C (17). According to Mašić et al. (26), the Digital Force Gauge device was utilized to assess the tibia bones' breaking strength. To determine the ash percentage, the bones of the tibia were subjected to a six-hour ashing in a muffle furnace set at 600°C. The composition of calcium and phosphorus in tibia ash was determined using a spectrophotometer (JEN WAY 3600). using a colorimetric method with BioVision kits© K380-200 and K410-500, respectively .

Gene expression

Data analysis with statistics

A one-way analysis of variance (ANOVA) was performed on all of the data. This data set was processed using SAS's GLM method (30). The means were separated using Duncan's multiple range tests (31), with significance being recognized at a level of P≤0.05.

Table 2: The forward and reverse primers for different genes

NaPi-Iia: sodium/phosphate type Iia; NaPi-Iib: sodium/phosphate type Iib; Calbindin: calcium binding protein

Results

Hen’s productive performance

Effect of NcaP on laying hens performance is presented in Table (3). Various amounts of NcaP considerably improved Hen’s performance metrics (P≤0.05) compared to control group (DCP). Most optimal (P≤0.05) egg yield, egg mass, and egg weight were recorded for birds have diets contained 50% NcaP. There were significant difference (P<0.05) in feed intake and FCR between NcaP groups vs. DCP group. Moreover, Raising NcaP levels resulted in a reduction (P≤0.05) in feed consumption. The FCR improved (P<0.05) by adding 50% NcaP level compared by other tested groups.  Whereas, the addition of NcaP in different levels gave the superior results compared to the control group.

Table 3: Effect of nano calcium phosphate on laying hens performance

 a, b, c,…. Etc. means in same raw within each factor  with different superscripts are significantly (P ≤ 0.05) different;     ^*significant at P≤0.05; P-value: probability value;  T1: 100%DCP; T2: 100%NcaP ; T3: 75%NcaP; T4: 50%NcaP; T5: 25%NcaP; DCP: dicalcium phosphate; NcaP: nano calcium phosphate             

Egg quality

The results herein indicated that supplementary NCaP enhanced egg quality and egg composition compared to DCP (Table 4). The addition of NCaP gave the superior (P≤0.05) eggshell thickness, weight, and percentage compared to the control group. In general, birds received NCaP at different substitution levels resulted in a higher (P≤0.05) eggshell thickness by 36.23- 40.11% than those received DCP group. Regarding the NCaP effect, yolk-albumin weight were markedly greater (P≤0.05) across all NCaP groups, except 25% NCaP group which gave the similar results with control group. The highest (P≤0.05) value of yolk-albumin weight (68.26g) was recorded for birds fed a diet contained 50% NCaP, while 25% NCaP and control groups recorded the lowest values (64.18 and 63.34g, respectively). Inclusion of dietary NCaP had no discernible change (P>0.05) in Haugh units, egg shape index, egg total lipids, total cholesterol, egg yolk color, egg albumin index and total cholesterol.

Blood biochemistry

As shown in Table (5), the experimental diet had no significant effect on tested blood parameters except for Ca and P concentrations but still within normal range (7-11 and 1.1-3.9 mmol/L for calcium and phosphorus, respectively) which are consistent with physiological values reported for laying hens during egg production (26). The blood Ca concentrations recorded significantly (P≤0.05) increased with NCaP groups when compared to DCP control group (T1). The group fed 100%NCaP was highest (P≤0.05) Ca and P concentrations among NCaP groups followed by 25% NCaP. Moreover, blood Ca and P amount decreased (P≤0.05) by decrease NCaP levels. These significant differences (P<0.05) in blood Ca and P values may be is related to different amount of daily feed consumed which Ca and P intake increased by levels of NCaP decreased in tested diets (Table 3), this relation reflect to Ca and P retained.

Sodium, potassium and, chloride concentrations had not significant differences between NCaP groups and DCP control group, although decreased values of those mineral with NCaP groups. The alkaline phosphatase (ALP), aspartate aminotransferase  (AST), glucose, and cholesterol values were not affected (P>0.05) due to NCaP dietary. Inaddition to no significant differences between all treatments  (P>0.05) in levels of Uric acid, total protein, albumin, globulin, and albumin / globulin ratio. The value of parathyroid hormone (PTH) recorded highest value (P>0.05) with 75 and 50% NCaP levels.

Table 4: Effect of nano calcium phosphate (NCaP) on egg quality and egg chemical analysis

a, b, c,…. etc. means in same raw within each factor  with different superscripts are significantly (P ≤ 0.05) different;  ^*significant at P≤0.05;  P-value: probability value;  T1: 100%DCP; T2: 100%NCaP ; T3: 75%NCaP; T4: 50%NCaP; T5: 25%NCaP; DCP: dicalcium phosphate; NCaP: nano calcium phosphate             

Figure 1 and 2 is illustrated effect various NCaP diets on the tibia bone properties of laying hens. Compared to the control group, tibia weight was considerably increased (P≤0.05) by inclusion NCaP levels. The birds which were fed 50% NCaP had the highest tibia weight (P≤0.05) and breaking strength of tibia (P>0.05). There was no changes (P>0.05) in the % tibia ash, calcium, or phosphorus content.

Figure 1: Tibia bone characteristics as affected by nanocalcium phosphate.

Figure 2: Tibia bone ash, calcium (Ca) and phosphorus (P) of laying hens as affected by nanocalcium phosphate.

Gene expression

The kidney's co-transporter sodium phosphate type II isoform a (NaPi-IIa) gene expression as affected (P≤0.05) by different sources of DCP (Figures 3). The addition of NCaP substantially raised (P≤0.05) the expression of NaPi-IIa gene compared to the control group (1.12), also hens given 50% NCaP exhibiting the highest expression (1.94). In the duodenum, the nano form recorded improved (P≤0.05) in NaPi-IIb, the best NaPi-IIb results (P≤0.05) were observed at 50% and 75% NCaP. In jejunum, all groups fed NCaP enhanced substantially (P≤0.05) more in NaPi-IIb gene expression compared to the DCP control group, with the highest (P≤0.05) effect observed in the 50% NCaP group (Figure 4).

The nano-form of minerals had an positive effect (P≤0.05) on the calbindin gene expression (calcium binding protein) in duodenum and jejunum of the gut. In the duodenum, calbindin gene expression was significantly (P≤0.05) improved with 100, 75, and 50%  of NCaP and the group provided the 50% NCaP had highest value (P≤0.05). In jejunum , the highest value (1.70; P≤0.05) of calbindin gene expression showed in 50% NCaP tested level group whereas, the DCP control group recorded lowest value (0.94; P≤0.05) of calbindin gene expression (Figure 5).

Table 5: Effect of nano calcium phosphate on blood biochemistry

a, b, c,…. etc. means in same raw within each factor  with different superscripts are significantly (P ≤ 0.05) different;     ^*significant at P≤0.05; P-value: probability value;  T1: 100%DCP; T2: 100%NCaP ; T3: 75%NCaP; T4: 50%NCaP; T5: 25%NCaP; DCP: dicalcium phosphate; NCaP: nano calcium phosphate; ALKP: alkaline phosphatase; AST: Aspartate transaminase; PTH: Parathyroid hormone.

Figure 3. NaPi-IIa gene expression in kidney of laying hens as affected by nanocalcium phosphate.

Figure 4: NaPi-IIb gene expression in intestine of laying hens as affected by nanocalcium phosphate.

Figure 5: Calbindin gene expression in intestine of laying hens as affected by nanocalcium phosphate.

Discussion

Hen’s productive performance parameters improved (P≤0.05) by NCaP levels were agreed with previous studies. Japanese quail' egg yield and weight affected (P≤0.05) due to NCaP supplementation either only or companied with DCP, whereas birds fed diet supplemented with 50% NCaP alone gave the best (P≤0.05) egg weight and production (19). The improvement in laying hen’s performance due to the very smaller particle size of the NCaP and more increase surface area than DCP, also NCaP improves the absorption through intestinal villi and increased bioavailability which led to enhanced productive performance (20). However, in terms of egg production, egg weight, egg mass, and FCR, no significant variations were noted in Bovans laying hens between Ca carbonate and nano-calcium carbonate (31). Generally, the groups fed 100, 75 and 50% NCaP were performed better than groups fed either control or 25% NCaP which almost had the same performance. The findings demonstrated that NCaP levels had a noticeable (P≤0.05) and beneficial effect on the performance of laying hens.

The current findings align with previous studies reporting improved eggshell quality following NCaP supplementation. For instance, inclusion 0.02% NCaP on shell thickness of brown LSL hen’s diet increased eggshell thickness by 11.24% over the control group. This attributed to impact of NCaP action on carbonic anhydrase enzyme activity, which takes part in eggshell formation and affects shell thickness (32). As noticed from the present results of the NCaP effect on egg quality, NCaP significantly increased, egg shell thickness, weight, and shell %. Nano Ca carbonate had improved the eggshell thickness of laying hens (31).

 Recently, dietary nanocalcium phosphate significantly (P≤0.05) enhanced the egg thickness of Japanese quail (33). Moreover, eggshell weight improved due to nano-selenium (21). Eggshell quality mainly depends on the bioavailability of Ca and P, as these two are essential minerals implicated in the formation of eggs and their components (34). However, Wang et al. (35) concluded that the particle size of Ca sources did not affect eggshell thickness or egg shape index. There is no effect of NCaP on the Haugh unit, which is a measure of albumin quality. In the present study, NCaP significantly improved shell thickness, shell weight, and shell percentage. These improvements may result from enhanced bioavailability of calcium and phosphorus, the major minerals responsible for shell formation and strength. However, similar to previous findings, NCaP supplementation did not significantly affect the Haugh unit, egg shape index, or albumen quality (36–38).

The present findings that NCaP increased serum Ca and P concentrations are consistent with previous reports. Ganjigohari et al. (37) observed that plasma calcium value decreased (P≤0.05) with decreased the level replacement of nanocalcium carbonate of laying hens blood. Makola et al. (14) recorded not significant differences between DCP and NCaP groups in ALP while, cholesterol concentration in broilers serum was significantly (P≤0.05) decreased.  However, serum Ca and P of LSL laying hens showed highest (P≤0.05) concentration with hens fed 400g NCaP /ton compared to control group and 200 and 800g NCaP/ton groups (38). This confirms that nano-sized calcium and phosphorus improve mineral absorption efficiency without disturbing blood homeostasis.

These findings demonstrate that nano form is superior to traditional form due to its smaller particle size and greater surface area, which enhance absorption and bioavailability (9, 39). Moreover, tibia bone characteristics (weight, length, breaking strength, and minerals content) more improved (P<0.05) by feeding nanominerals (6-7, 16). Adding of inorganic sources to poultry feeds lead to lower mineral bioavailability compared to organic sources, increasing productive performance and bone quality was achieved by low dosages from nano Ca and P (40). However, adding nano calcium carbonate to laying hen diets increased their tibia bone weight and thickness, but had no effect on tibia ash % (41).

The NaPi-IIa was measured to observe the reabsorption of P in kidney. The results observed that  NCaP reduced P excretion by increase the expression of NaPi-IIa in the apical brush border of the renal proximal tubule epithelial cells, which is renal sodium phosphate cotransporter responsible for reabsorption of P from the urine to the blood so reducing the mineral excretion (42-43).

The present findings demonstrated that NCaP decreased P excretion. The NaPi-IIb cotransporter is an essential component for the active transport of P across intestinal epithelial cells (40). Intestinal duodenum and jejunum P absorption was improved with the addition of NCaP than the control. The active transport of Ca through the intestines is facilitated by calbinidin, a protein that binds Ca (41). The NCaP feeding increased intestinal calcium absorption by increasing calbinidin expression within the birds duodenum and jejunum compared to those fed the traditional form. The use of small amounts of NCaP improved productivity and reduced the excretion of Ca and P by approximately half (15). A reduction excretory Ca and P concentrations by 50% as a result of utilization lower nano mineral dosages (18). Moreover, dietary DCP and nano P levels increased in chick diets , the quantity of mRNA levels of NaPi-IIb decreased in the gut (14). Diets low Ca content enhanced duodenal expression of calpindin and NaPi-IIb mRNA transporters (44-45).

Nanoparticles confer advantageous effects in poultry via several physiological, biochemical, and cellular pathways that improve food utilization, bolster antioxidant status, and boost immunity while preserving gut health (46-50).

Improved Intestinal Absorption and Bioavailability: The nanoscale dimensions of calcium phosphate particles (often <100 nm) augment their surface area and solubility within the gastrointestinal tract. This enhances ionization and interaction with intestinal transporters (such as Ca²⁺ and PO₄³⁻ channels), resulting in increased absorption in the duodenum and jejunum. Thus, the efficiency of calcium and phosphorus utilization is superior compared to that of birds consuming standard dicalcium phosphate (DCP) (51-53).

Enhanced Bone Mineralization and Skeletal Integrity: Upon absorption, NCaP provides accessible calcium and phosphorus for the synthesis of hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂), the principal mineral constituent of bone. The nanoscale form enhances the calcium-to-phosphorus ratio at the cellular level, facilitating improved osteoblast activity and collagen cross-linking. This leads to denser, stronger bones with enhanced tibia ash, breaking strength, and bone mineral density, hence diminishing the incidence of leg diseases and fractures in broilers and layers (54-56).

Improvement of Eggshell Quality (in Layers): NCaP offers a consistent and bioavailable source of Ca²⁺ ions essential for shell calcification in the shell gland (uterus). The enhanced calcium deposition augments shell thickness, strength, and specific gravity, hence diminishing the incidence of cracked or soft-shelled eggs. Moreover, adequate phosphorus availability inhibits calcium transport from bones, hence preserving skeletal reserves and prolonging laying persistency  (57-58).

Cellular and Molecular Mechanisms: NCaP stimulate calcium-sensing receptors and improve intracellular signaling (e.g., via MAPK and PI3K/Akt pathways) via interacting with intestinal epithelial cells and osteoblast membranes. Alkaline phosphatase (ALP) and osteocalcin, two bone matrix proteins necessary for mineral deposition and bone remodeling, are upregulated as a result. By preserving redox equilibrium, NCaP also lessens oxidative stress in intestinal and bone tissues, promoting tissue integrity and cell survival (48, 59-63).

Environmental and Nutritional Efficiency: Due to its elevated absorption rate, dietary NCaP permits reduced phosphorus doses while preserving performance and bone integrity. This reduces phosphorus excretion and lowers environmental pollution, hence promoting sustainable poultry production (33, 54).

Conclusion          

The incorporation of NCaP in laying hen diets significantly enhanced productive performance and improved egg quality. It increased the calcium and phosphorus retention, resulting in stronger eggshells and superior bone mineralization. Moreover, enhanced mineral absorption resulted in less excretion, so aiding in the reduction of environmental contamination. NCaP provides an effective and sustainable approach to enhancing poultry health, productivity, and egg quality.

Acknowledgment               

The authors would like to express their sincere gratitude to Cairo University of Egypt for its support and valuable facilities that contributed significantly to the success of this research.

Conflict of interest

No authors have disclosed any potential bias or conflict of interest.