Calcium pidolate supplementation enhances broiler growth, bone strength, and mineral utilization under reduced dietary calcium and phosphorus levels

Abstract

This study aimed to evaluate whether calcium pidolate supplementation can mitigate the negative impacts of calcium (Ca) and available phosphorus (aP) restriction in broiler diets. Eight hundred one-day-old broiler chicks were randomly assigned to five treatments (8 replicates/treatment): control diet (CONT); 15% reduced Ca and aP diet (N15); N15 supplemented with 300 mg/kg calcium pidolate (CP15); 30% reduced Ca and aP diet (N30); and N30 with 300 mg/kg calcium pidolate (CP30). Broilers were reared for 35 days, with growth performance recorded weekly. Mineral metabolism indicators were analyzed [parathyroid hormone (PTH), Fibroblast growth factor 23 (FGF23) and alkaline phosphatase (ALP)], as well as tibia quality, carcass traits, nutrient digestibility, and litter characteristics. Data were analyzed using one-way ANOVA in SPSS. The CP15 results achieved the highest final BW among treatments (P < 0.05) and lower FCR than N15, but similar to CONT. Birds fed the CP15 diet maintained serum Ca and phosphorus levels and bone ash percentage similar to CONT, while also exhibiting lower PTH levels than N15 (P < 0.05); however, it had similar FGF23 and ALP levels as CONT and N15. In contrast, the N30 markedly impaired (P < 0.05) BW, FCR and bone ash compared to CP15. For CP30, growth performance was similar to that of CONT and better than that of N15 and N30. Ash and phosphorus digestibility were similar to N30, and were higher than N15 and CONT groups. Although bone ash and phosphorus mineralization were similar to CONT and CP15 groups and higher than N30, bone Ca mineralization was the lowest for CP30 and N30 groups. Physical tibia parameters and litter characteristics were not affected by treatments. Carcass traits were similar among treatments, except that breast meat yield was maintained in CP15 but was significantly reduced in N30 and CP30 groups. In conclusion, calcium pidolate supplementation optimizes mineral utilization and preserves broiler production efficiency during mineral-restricted feeding programs.

Introduction

Calcium (Ca) and phosphorus (P) are essential nutrients in broiler diets, required not only for skeletal development but also for muscle function and metabolic processes. Extensive research has established minimum dietary requirements for growth and bone integrity (NRC, 1994; Qian et al., 1997; Angel, 2013), forming the basis of current nutritional guidelines (David et al., 2021; Reis et al., 2023; Lee, 2026). Because Ca and P interact at multiple levels—absorption, metabolism, and deposition in bone—their dietary balance is critical for optimal production (Rousseau et al., 2016; Angel, 2019; Woyengo et al., 2022).

Calcium and P dietary imbalance has a detrimental effect on broilers productivity (Gautier et al., 2017). When dietary Ca is fed in excess, it binds to phytate in the gastrointestinal tract, forming insoluble complexes that reduce P solubility and its absorption, interfere with fat emulsification (Abdulla et al., 2017), and decrease productivity (Valable et al., 2017; Hakami et al., 2022). When dietary Ca and P are deficient, skeletal integrity of birds and growth performance are also compromised (Wu et al., 2023). For instance, Rousseau et al. (2016) found that feeding Ca and P-deficient diets reduced ADG by up to 13% and tibia ash by up to 9% in broiler chickens. Therefore, a balanced mineral nutrition is crucial to support efficient FCR and lean tissue accretion (Hakami et al., 2022; Imari et al., 2022).

At formulation level, calcium is one of the least expensive components in broiler diets; however, when supplied in excess, it can act as an antinutritional factor and impair gut health (Walk et al., 2021; David et al., 2023). Phosphorus, by contrast, is expensive, finite, and environmentally contaminant due to its high excretion rates (Abdelhady et al., 2015; Chakraborty et al., 2021). These economic and environmental concerns have made reducing dietary Ca and P an important goal for sustainable poultry production. Nevertheless, the rapid growth rates and muscle deposition of modern broiler strains necessitate more precise mineral management to maintain skeletal integrity and performance (Aviagen, 2022).

Several nutritional strategies have been proposed to reduce Ca and P inclusion without impairing performance. These include depletion–repletion programs that stimulate adaptive mineral absorption (Valable et al., 2017), the use of low-solubility Ca sources, and higher phytase dosages to minimize dietary non-phytate P (Hamdi et al., 2015; Gautier et al., 2017). While such approaches help, novel mineral sources may further optimize utilization.

Organic sources of Ca, like calcium pidolate, also stand as an efficient strategy to support Ca and P balance in poultry. Several studies reported its efficiency in supporting egg production and eggshell quality in laying hens and laying ducks (Agblo and Duclos, 2012; Valderrama and Roulleau, 2013; Bain et al., 2018; Sarmiento-García et al., 2022) and optimizing bone mineralization, Ca digestibility, meat quality, muscle myopathies and growth performance of broilers (Fondevila et al., 2021; Iglesias et al., 2023; Aguilon et al., 2024; Bertechini et al., 2024; Gul, 2024). Calcium pidolate consists of a calcium salt of pyroglutamic acid. Its proposed mode of action involves the stimulation of specific protein synthesis via arginine pathways (Joshi et al., 2019; Bertechini et al., 2024), which enhances the intestinal absorption and systemic bioavailability of Ca and P. Furthermore, pyroglutamic acid acts as a precursor for collagen synthesis, thereby improving Ca and P retention within the bone matrix (Price, 2012; Aguilon et al., 2024). Therefore, calcium pidolate offers a targeted metabolic solution to Ca and P challenges and is suggested to improve their metabolic balance in birds (Fondevila et al., 2021). Furthermore, its utilization could optimize dietary mineral levels and provide more precise nutrition (Fondevila et al., 2021). We hypothesize that supplementing calcium pidolate to a Ca and P deficient broilers diet might sustain growth and productivity without affecting bone reserves and metabolic processes.

The objective of this study was to determine whether calcium pidolate supplementation can sustain or improve growth performance, mineral balance, bone quality, nutrient digestibility, and carcass traits in broilers fed diets with Ca and available phosphorus (aP) below the requirements.

Materials and methods

The experiment was conducted at the Poultry Experimental Farm of the Faculty of Agriculture of Ain Shams University, located in Shalaqan Village, Qalyubia Governorate, Egypt. All procedures followed ethical guidelines approved by the Institutional Animal Care and Use Committee (approval no. 5/2024/28).

Birds and management practices

A total of 800 one-day-old mixed-sex Arbor Acres broiler chicks (as hatched) were obtained from a commercial hatchery and vaccinated post-hatch against Newcastle disease virus and infectious bronchitis virus. Standard poultry management practices and vaccination protocols were strictly followed. The chicks were reared under a controlled lighting schedule, starting with 23 hours of light for the first three days, followed by 20 hours of light and 4 hours of darkness from days 4 to 12, and finally 18 hours of light and 6 hours of darkness from days 13 to 35. During the first week, house temperature was maintained between 34°C and 31°C and subsequently reduced gradually by approximately 3°C per week until reaching 26°C. Chicks were observed daily for overall flock health, temperature control, lighting, feed, water availability, and any unexpected occurrences. The birds were raised in floor pens (1.5 m² each) on wood-shaving litter under uniform management conditions. They received feed and water ad libitum, using a commercial corn-soy-based diet. Mortality was monitored twice daily, and dead chicks were immediately removed and recorded.

Experimental design and diets

The chicks were randomly allocated to five dietary treatment groups in a complete randomized design. Each treatment was replicated 8 times, with 20 chicks per pen. The treatments consisted of: 1) control group animals (CONT) received a basal diet formulated according to FEDNA (2021) nutritional requirements for unsexed broilers (Table 1a, Table 1b, Table 1c), provided in three phases: starter (0–14 days) formulated to 1.01% Ca and 0.45% aP, grower (15–27 days) formulated to 0.92% Ca and 0.40% aP, and finisher (28–35 days) formulated to 0.8% Ca and 0.34% aP; 2) N15 group animals, fed a diet with 15% lower Ca and aP compared to the CONT diet; 3) CP15 group, consisted of N15 + calcium pidolate at 300 mg/kg (CALPID, Norel Animal Nutrition); N30 group animals, fed a diet with 30% lower Ca and aP compared to the CONT diet; and 4) CP30, consisted of N30 + calcium pidolate at 300 mg/kg. The calcium pidolate dose was selected based on the manufacturer’s commercial recommendations for broiler chickens. This dosage of calcium pidolate had been previously tested in other studies by Fondevila et al. (2021) and Iglesias et al. (2023). All diets were pelleted and analyzed for dry matter (ISO 6496), ash (ISO 5984), crude protein (ISO 5983), P (ISO 6491:1998), and Ca (ISO 6869).

Table 1a.

Ingredient composition and analyzed nutrient content of experimental diets during the (A) starter (0-14 d) phases.

Ingredient	STARTER (0-14 d)
	CONT	N15	CP15	N30	CP30
Yellow Corn	545.50	558.80	558.80	566.00	566.00
Soybean Meal (46%)	330.50	329.95	329.95	329.89	329.89
Corn Gluten Meal (60%)	41.00	40.58	40.58	39.46	39.46
Wheat middling	21.00	18.64	18.64	21.51	21.51
Soybean Oil	18.90	15.12	15.12	12.12	12.12
Calcium Carbonate	18.37	15.99	15.99	13.63	13.63
Monocalcium phosphorus	10.17	6.62	6.62	3.03	3.03
CALPID	0.00	0.00	0.30	0.00	0.30
Salt (NaCl)	4.29	4.31	4.31	4.33	4.33
HCL Lysine	3.46	3.45	3.45	3.43	3.43
DL Methionine	1.71	1.71	1.71	1.71	1.71
L-Threonine	0.81	0.81	0.81	0.81	0.81
Choline chloride 60%	0.50	0.50	0.50	0.50	0.50
Coccidiostat	0.30	0.30	0.30	0.30	0.30
Phytase enzyme	0.10	0.10	0.10	0.10	0.10
NSPase Enzyme	0.10	0.10	0.10	0.10	0.10
Vitamins and minerals premix1	3.00	3.00	3.00	3.00	3.00
Calculated composition2
Crude Protein, %	22.3	22.3	22.3	22.32	22.32
ME, kcal/kg	2950	2950	2950	2948	2948
Crude fat, %	4.4	4.09	4.09	3.82	3.82
Crude fiber, %	3.20	3.21	3.21	3.25	3.25
Linoleic acid, %	2.21	2.02	2.02	1.88	1.88
Calcium, %	1.01	0.85	0.85	0.71	0.71
Available P, %	0.45	0.38	0.38	0.32	0.32
dig Lysine, %	1.22	1.22	1.22	1.22	1.22
dig Methionine, %	0.5	0.5	0.5	0.5	0.5
dig TSAA, %	0.78	0.79	0.79	0.79	0.79
Analyzed chemical Composition
Moisture	11.32	11.25	11.38	11.33	11.42
Dry matter	88.68	88.75	88.62	88.67	88.58
ASH	5.99	5.38	5.35	5.01	5
Crude Protein %	22.49	22.57	22.49	22.41	22.52
Calcium %	1.08	0.94	0.93	0.77	0.75
Phosphorus %	0.67	0.59	0.59	0.48	0.47

¹Each 3 Kg of premix contains: Vitamins: A: 12000000 IU; Vit. D3 5000000 IU; E: 10000 mg; K3: 2000 mg; B1:1000 mg; B2: 5000 mg; B6:1500 mg; B12: 10 mg; Biotin: 50 mg; Pantothenic acid: 10000 mg; Nicotinic acid: 30000 mg; Folic acid: 1000 mg; Minerals: Mn: 60000 mg; Zn: 50000 mg; Fe: 30000 mg; Cu: 10000 mg; I: 1000 mg; Se: 100 mg and Co: 100 mg.

²Calculated according to FEDNA 2021. All diets were provided in Pellet form on an ad libitum basis.

Table 1b.

Ingredient composition and analyzed nutrient content of experimental diets during the (B) grower (15-27 d), phases.

Ingredient	GROWER (15-27 d)
	CONT	N15	CP15	N30	CP30
Yellow Corn	573.90	585.20	585.20	596.50	596.50
Soybean Meal (46%)	173.45	172.91	172.91	172.36	172.36
Corn Gluten Meal (60%)	100.00	100.00	100.00	100.00	100.00
Wheat middling	89.77	87.91	87.91	86.05	86.05
Soybean Oil	20.56	17.03	17.03	13.50	13.50
Calcium Carbonate	17.94	15.75	15.75	13.56	13.56
Monocalcium phosphorus	8.23	5.07	5.07	1.91	1.91
CALPID	0.00	0.00	0.30	0.00	0.30
Salt (NaCl)	4.08	4.10	4.10	4.11	4.11
HCL Lysine	5.59	5.60	5.60	5.61	5.61
DL Methionine	1.27	1.14	1.14	1.12	1.12
L-Threonine	1.15	1.26	1.26	1.25	1.25
Choline chloride 60%	0.50	0.50	0.50	0.50	0.50
Coccidiostat	0.30	0.30	0.30	0.30	0.30
Phytase enzyme	0.10	0.10	0.10	0.10	0.10
NSPase Enzyme	0.10	0.10	0.10	0.10	0.10
Vitamins and minerals premix1	3.00	3.00	3.00	3.00	3.00
Calculated composition2
Crude Protein, %	20.16	20.18	20.18	20.21	20.21
ME, kcal/kg	3050	3050	3050	3050	3050
Crude fat, %	4.82	4.50	4.50	4.18	4.18
Crude fiber, %	3.30	3.30	3.30	3.30	3.30
Linoleic acid, %	2.42	2.24	2.24	2.06	2.06
Calcium, %	0.92	0.78	0.78	0.64	0.64
Available P, %	0.40	0.34	0.34	0.28	0.28
dig Lysine, %	1.09	1.09	1.09	1.09	1.09
dig Methionine, %	0.45	0.45	0.45	0.45	0.45
dig TSAA, %	0.73	0.73	0.73	0.73	0.73
Analyzed chemical Composition
Moisture	10.31	10.24	10.32	10.54	10.27
Dry matter	89.69	89.76	89.68	89.46	89.73
ASH	5.25	4.74	4.75	4.22	4.31
Crude Protein %	20.45	20.50	20.37	20.40	20.43
Calcium %	0.94	0.79	0.77	0.67	0.67
Phosphorus %	0.57	0.50	0.49	0.41	0.40

¹Each 3 Kg of premix contains: Vitamins: A: 12000000 IU; Vit. D3 5000000 IU; E: 10000 mg; K3: 2000 mg; B1:1000 mg; B2: 5000 mg; B6:1500 mg; B12: 10 mg; Biotin: 50 mg; Pantothenic acid: 10000 mg; Nicotinic acid: 30000 mg; Folic acid: 1000 mg; Minerals: Mn: 60000 mg; Zn: 50000 mg; Fe: 30000 mg; Cu: 10000 mg; I: 1000 mg; Se: 100 mg and Co: 100 mg.

²Calculated according to FEDNA 2021. All diets were provided in Pellet form on an ad libitum basis.

Table 1c.

Ingredient composition and analyzed nutrient content of experimental diets during the (C) finisher (28-35 d) phases.

Ingredient	FINISHER (28-35 d)
	CONT	N15	CP15	N30	CP30
Yellow Corn	605.80	615.90	615.90	625.80	625.80
Soybean Meal (46%)	205.99	205.96	205.96	205.93	205.93
Corn Gluten Meal (60%)	73.22	72.47	72.47	71.74	71.74
Wheat middling	54.60	52.83	52.83	51.10	51.10
Soybean Oil	25.69	22.72	22.72	19.82	19.82
Calcium Carbonate	16.00	14.06	14.06	12.05	12.05
Monocalcium phosphorus	5.18	2.50	2.50	0.00	0.00
CALPID	0.00	0.00	0.30	0.00	0.30
Salt (NaCl)	3.85	3.87	3.87	3.88	3.88
HCL Lysine	3.86	3.86	3.86	3.86	3.86
DL Methionine	0.94	0.94	0.94	0.94	0.94
L-Threonine	0.63	0.63	0.63	0.63	0.63
Choline chloride 60%	0.50	0.50	0.50	0.50	0.50
Coccidiostat	0.50	0.50	0.50	0.50	0.50
Phytase enzyme	0.10	0.10	0.10	0.10	0.10
NSPase Enzyme	0.10	0.10	0.10	0.10	0.10
Vitamins and minerals premix1	3.00	3.00	3.00	3.00	3.00
Calculated composition2
Crude Protein, %	19.50	19.50	19.50	19.50	19.50
ME, kcal/kg	3100	3100	3100	3100	3100
Crude fat, %	5.30	5.03	5.03	4.76	4.76
Crude fiber, %	3.10	3.10	3.10	3.10	3.10
Linoleic acid, %	2.68	2.53	2.53	2.39	2.39
Calcium, %	0.80	0.68	0.68	0.56	0.56
Available P, %	0.34	0.29	0.29	0.24	0.24
dig Lysine, %	1.00	1.00	1.00	1.00	1.00
dig Methionine, %	0.41	0.41	0.41	0.41	0.41
dig TSAA, %	0.68	0.68	0.68	0.68	0.68
Analyzed chemical Composition
Moisture	11.02	11.10	11.08	10.96	10.94
Dry matter	88.98	88.90	88.92	89.04	89.06
ASH	4.10	3.89	3.88	3.49	3.50
Crude Protein %	19.18	19.21	19.15	19.22	19.15
Calcium %	0.79	0.65	0.63	0.55	0.54
Phosphorus %	0.43	0.38	0.39	0.32	0.33

¹Each 3 Kg of premix contains: Vitamins: A: 12000000 IU; Vit. D3 5000000 IU; E: 10000 mg; K3: 2000 mg; B1:1000 mg; B2: 5000 mg; B6:1500 mg; B12: 10 mg; Biotin: 50 mg; Pantothenic acid: 10000 mg; Nicotinic acid: 30000 mg; Folic acid: 1000 mg; Minerals: Mn: 60000 mg; Zn: 50000 mg; Fe: 30000 mg; Cu: 10000 mg; I: 1000 mg; Se: 100 mg and Co: 100 mg.

²Calculated according to FEDNA 2021. All diets were provided in Pellet form on an ad libitum basis.

Performance evaluation

Pen BW and feed intake (FI) were recorded at 1, 7, 14, 21, 28, and 35 days of age. Feed intake was adjusted for mortality by accounting for the weight of dead birds to calculate the feed conversion ratio (FCR) at the end of the trial (day 35). The European Production Efficiency Factor (EPEF) was calculated using the formula:

EPEF = (live body weight (kg) × livability) / (age in days × FCR) × 100.

Blood samples and determination of plasma biochemicals

One bird per pen was randomly selected for blood, tibia and carcass analysis at 35days. Blood samples were collected immediately at slaughter and centrifuged at 3,000 rpm for 15 min to separate plasma and serum. The PTH concentrations were determined using an intact PTH ELISA kit (Immutopics, USA; Cat. No. 60-2500). The FGF23 concentrations were measured using ELISA kits (Cusabio®, China; Cat. No. CSB-E12170R-1). Serum Ca, P, and alkaline phosphatase (ALP) activity were determined colorimetrically using bioanalyzer diagnostic test kits (SPINREACT, Santa Coloma, Spain) with a spectrophotometer (Jenway, Model 6850 UV/Visible double beam). All analyses were performed according to the manufacturer’s instructions.

Carcass characteristics

The remaining carcass from the same birds used for blood sample analysis were weighed and expressed as a percentage of live body weight (LBW), while internal organs like liver, pancreas, spleen, and abdominal fat were weighed and expressed as a percentage of LBW.

Tibia bone sampling and measurements

Both tibia bones from each of the previous birds were collected and thoroughly cleaned of soft tissues and cartilage. The wet tibia weight was measured relative to the birds LBW to calculate the wet tibia weight percentage. Bone chemistry analysis was conducted on the left tibia samples; both dried bone and feed samples were ashed for 4 hours at 650°C, and the resulting ash was dissolved in hydrochloric acid. An Atomic Emission Spectrometer was then used to determine the concentrations of dietary Ca and P, as well as the P content in the bone according to Abdelaziz et al. (2019).

The right tibia bones were used for physical measurements. Tibia length (in mm) and tibia shaft diameter (in mm) were measured using a digital caliper with 0.01 mm accuracy according to the method described by Samejima (1990). Additionally, the tibia Seedor index, which reflects tibia mineral density, was calculated, and the maximum breaking force was determined following the procedure described by Seedor et al. (1991). In parallel, bones from the right drumsticks were subjected to colorimetric analysis using a Handheld Colorimeter - Precise Color Analyzer Digital Color Reader with an 8 mm probe mounted on the proximal epiphysis. The lightness (L*), redness (a*), and yellowness (b*) of the tibia were measured, with three readings taken per epiphysis and averaged for statistical analysis (El-Safty et al., 2022).

Nutrient digestibility

At 35 days, five broiler chicks per treatment were used for a 3-day digestibility trial following a 12-hour fast. Feed intake was monitored and excreta were collected over 3 days. Excreta were treated with 4% boric acid, weighed, and dried at 60°C until a constant weight. The dried samples were ground and stored for analysis. Apparent digestibility coefficients were calculated as: [(Nutrient intake – Nutrient excretion) / Nutrient intake] x 100. Organic matter, Ca, and P were determined according to AOAC methodology (2002) and expressed on a dry matter basis.

Litter characteristics

At days 21 and 35, four fresh samples of 250 g were randomly collected from the full depth of each pen and pooled and mixed in one composite sample per pen. Moisture content and pH were measured in five samples per treatment group. Afterward, samples were placed in sealed bags and immediately frozen. The percentage of collected samples was computed as: (weight of dried sample / fresh weight used) x 100 (Ezenwosu et al., 2022). Mineral concentrations of Ca and P were subsequently determined using inductively coupled plasma spectroscopy according to AOAC (2002).

Statistical analysis

Data were analyzed using one-way ANOVA for a completely randomized design using the GLM procedure in SPSS software (version 20.0). Data normality and homogeneity were assessed by Shapiro-Wilk and Levene’s tests, respectively. Each pen containing 20 chickens (or the animals sampled from those pens) served as the experimental unit. Differences among treatment means were identified using Tukey’s Honestly Significant Difference (HSD) test. The statistical model applied was: Yij = μ + Ti + eij, where: Yij = observation of measured parameter, μ = overall mean, Ti = effect of treatment (i: 1 to 5), eij = random experimental error. Differences were considered significant when P ≤ 0.05.

Results

Animal performance parameters

The effects of dietary treatments on broiler performance are shown in Table 2. Growth performance was affected by dietary treatment for the different nutritional phases (P < 0.05). Initial chick weights were similar among treatment groups (average 43.41 g). At day 14, CP15 had the highest LBW, followed by CONT and CP30, while N30 and N15 had the lowest LBW. At the end of the grower phase, CP15 continued to have the highest LBW, followed by CONT and then N15 and CP30, while the N30 group had the lowest LBW. At the end of the trial at 35 days, the CP15 group achieved the highest LBW among treatments, followed by CONT and CP30. The N30 group had the lowest LBW at the end of the trial. For FI, CP15 had the highest FI during the 3 nutritional phases of the trial, while the CONT and N15 groups were intermediate and N30 and CP30 had the lowest FI. The overall FCR of the trial was better in the CP15, CONT and CP30 groups, and was poorer in the N15 and N30 groups. The EPEF index showed the highest value for the CP15 group and the lowest for the N30 group (P < 0.001). The CP30 group exhibited an intermediate value and was similar to the CONT and N15 groups.

Table 2.

Effect of dietary treatments on productive performance and European Production Efficiency Factor of broiler chicks from 1 to 35 days.

Treatment	CONT	N15	CP15	N30	CP30	SEM	P-value
Live body weight, g
Initial weight	43.54	43.53	43.06	43.28	43.66	0.086	0.183
7 days	200a	197b	201a	196b	199ab	0.592	0.011
14 days	533b	517c	548a	507c	529b	17.54	<0.001
21 days	999a	960b	1002a	925c	963b	29.86	<0.001
28 days	1546b	1512c	1615a	1451d	1502c	9.94	<0.001
35 days	2013b	1920c	2083a	1876d	1925b	13.20	<0.001
Feed Intake, g
0-7 day	156c	160b	156c	165a	156c	0.701	<0.001
8-14 day	431ab	421b	442a	407c	420b	18.37	<0.001
15-21 day	704c	703c	685a	661b	664b	20.62	<0.001
22-28 day	1027bc	1062b	1129a	1035bc	1014c	8.76	<0.001
29-35 day	898a	848c	895a	892a	877b	5.80	0.027
Overall	3216b	3194c	3306a	3160cd	3130d	10.850	<0.001
Feed conversion ratio (FCR), g:g
0-7 day	0.99c	1.05b	0.99c	1.08a	1.00c	0.007	<0.001
8-14 day	1.30a	1.31a	1.27b	1.31a	1.27b	0.02	<0.001
15-21 day	1.51b	1.59a	1.51b	1.58a	1.53b	0.04	<0.001
22-28 day	1.88bc	1.92b	1.84c	1.97a	1.88bc	0.01	<0.001
29-35 day	1.93b	2.07ab	1.91b	2.10a	2.07a	0.01	<0.001
Overall	1.63b	1.70a	1.62b	1.72a	1.66b	0.018	<0.001
European Production Efficiency Factor (EPEF)
EPEF	330ab	304cd	342a	287d	310bc	3.092	<0.001

^ᵃ-ᵈMeans within a row with no common superscript differ significantly (p < 0.05).

EPEF = [Liveability (%) × Live weight (kg)] / (Age (d) × FCR) × 100]. Values are presented for informational purposes and were not subjected to statistical analysis.

Serum biochemical parameters

Blood parameters at day 35 are shown in Table 3. Dietary treatment affected all measured metabolites (P < 0.001). The PTH and FGF23 levels were the highest in N30, followed by CP30 and N15, while CONT and CP15 maintained the lowest levels. Serum Ca and P were the highest in the CONT and CP15 groups, while the N15 group had the lowest Ca level and the N30 group had the lowest P level. The ALP activity was the highest in N30 and CP30 and the lowest in CONT.

Table 3.

Effect of the dietary supplementation of calcium pidolate on the blood parameters of broilers in each experimental group at 35 days.

Parameters1	CONT	N15	CP15	N30	CP30	SEM	P-value
PTH, (pg/mL)	7.87c	10.51b	8.56c	13.28a	9.43bc	0.394	<0.001
FGF23, (pg/mL)	81.51d	100.72bc	89.47bc	115.64a	106.38ab	2.625	<0.001
Ca, (mg/dL)	9.840a	8.530c	9.830a	9.200b	9.320b	0.483	<0.001
P, (mg/dL)	4.490a	4.370b	4.500a	4.050c	4.500a	0.173	<0.001
ALP, (U/L)	174.28b	187.23ab	187.35ab	192.16a	197.07a	7.591	<0.001

^a–cValues within a row with different superscripts differ significantly (P < 0.05).

¹PTH = parathyroid hormone; FGF23 = fibroblast growth factor 23; Ca = calcium; P = phosphorus; ALP = alkaline phosphatase.

CONT = control diet formulated to meet Ca and aP requirements; N15 = negative control diet with 15% lower Ca and aP than CONT; CP15 = N15 supplemented with 300 mg/kg calcium pidolate; N30 = negative control diet with 30% lower Ca and aP than CONT; CP30 = N30 supplemented with 300 mg/kg calcium pidolate.

SEM = standard error of the mean (overall). n = 8 birds per treatment.

Carcass characteristics

Carcass traits expressed as a percentage of LBW of broilers were not different among treatments, except for breast yield (P < 0.001, Table 4). Breast yield was the lowest in the N30 and CP30 groups compared to the CONT, N15, and CP15 groups, where no difference was observed among them. The thigh, drumstick, abdominal fat, liver, gizzard, heart, and spleen percentages remained unaffected.

Table 4.

Effect of the dietary supplementation of calcium pidolate on the selected carcass traits (expressed as % of live body weight) of broilers in each experimental group.

Treatment	CONT	N15	CP15	N30	CP30	SEM	P-value
Dressing1, %	71.35	71.00	71.10	69.73	69.31	0.286	0.076
Breast2,3, %	32.72a	32.40a	31.98a	28.67b	28.63b	0.316	<0.001
Thigh3, %	14.88	14.74	14.78	15.04	14.88	0.094	0.894
Drumstick3, %	8.37	8.66	8.53	8.34	8.39	0.068	0.546
Abdominal fat, %	2.76	2.83	2.93	2.71	2.38	0.067	0.095
Liver, %	2.53	2.41	2.48	2.56	2.35	0.028	0.464
Gizzard, %	1.28	1.29	1.26	1.32	1.32	0.006	0.962
Heart, %	0.40	0.41	0.41	0.40	0.40	0.002	0.950
Spleen, %	0.09	0.09	0.09	0.09	0.09	0.002	0.284

^aand ^b Values with different superscripts in the same row were significantly different (p < 0.05). CONT: control diet formulated to meet Ca and aP requirements; N15: negative control diet reformulated to 15% lower Ca and aP compared CONT; CP15: negative control diet reformulated to 15% lower Ca and aP compared to the CONT supplemented with 300 mg/kg of calcium pidolate; N30: Negative control diet reformulated to 30% lower Ca and aP compared CONT; CP30: Negative control diet reformulated to 30% lower Ca and aP compared CONT supplemented with 300 mg/kg of calcium pidolate. SEM: Standard error of the mean (overall), n = 8 birds per treatment.

¹Eviscerated carcass (with neck, abdominal fat).

²With wings, skin, and bones.

³With skin and bones.

Tibia bone measurements

Tibia bone physical measurements (dry weight, length, diaphysis diameter, and volume) of birds were not affected by the dietary treatments (P > 0.05); however, the chemical content of the tibia was different among treatments (P < 0.05, Table 5). Birds in the CONT and CP15 groups had the highest ash content compared to the N15 and N30 groups, while the CP30 group ash content remained intermediate. The CONT group had the highest tibia Ca level, and the N30 and CP30 had the lowest, while the CP15 Ca level was not different than the CONT group. The tibial P content was the highest in the CONT, CP15 and CP30 groups and the lowest in the N30 group, while the N15 group exhibited an intermediate value (P = 0.054). The TBS followed a similar trend, with N30 birds showing lower strength compared to the CONT and CP15. The tibia Seedor index remained relatively stable across treatments, reflecting no major differences in overall bone density. Bone color (lightness, redness, and yellowness) was not affected by dietary treatments.

Table 5.

Effect of the dietary supplementation of calcium pidolate on the tibia bone measurements of broilers in each experimental group at 35 days.

Parameters		CONT	N15	CP15	N30	CP30	SEM	P-value
Dry Weight (%) from LBW		0.530	0.540	0.530	0.540	0.540	0.005	0.182
Length, (mm)		91.310	91.250	91.880	91.070	90.810	0.354	0.102
Diaphysis diameter, (mm)		6.260	6.180	6.320	6.170	6.210	0.056	0.060
Volume, (cm3)		8.250	8.140	8.430	8.450	8.400	0.120	0.300
Seedor index, (g/mm)⁎⁎		111.05	110.66	111.32	110.52	112.48
Ash, (%)		47.430a	44.150b	47.140a	43.510b	45.930ab	0.410	<0.001
Ca, (%)		19.060a	17.230b	18.970ab	16.240c	16.970c	0.230	<0.001
P, (%)		10.460a	10.090ab	10.590a	9.500b	10.300a	0.120	0.054
TBS, (kg)***		28.330a	25.310b	29.010a	26.190b	27.670ab	0.390	<0.001
Color⁎⁎⁎⁎	L* lightness	26.260	25.182	25.650	25.088	25.305	0.557	0.970
	a* redness	1.883	2.260	1.918	2.607	2.077	0.141	0.497
	b* yellowness	9.236	9.738	9.925	10.737	8.645	0.323	0.334

^a-cvalues with different superscripts in the same row were significantly different (p < 0.05).

CONT: control diet formulated to meet Ca and aP requirements; N15: negative control diet reformulated to 15% lower Ca and aP compared CONT; CP15: negative control diet reformulated to 15% lower Ca and aP compared CONT supplemented with 300 mg/kg of calcium pidolate; N30: Negative control diet reformulated to 30% lower Ca and aP compared to the CONT; CP30: Negative control diet reformulated to 30% lower Ca and aP compared to the CONT supplemented with 300 mg/kg of calcium pidolate. SEM: Standard error of the mean (overall), n = 8 birds per treatment.

^⁎⁎Seedor index described by Seedor et al. (1991): TBS: Tibia Breaking Strength;.

^⁎⁎⁎⁎color was determined by using a Handheld Colorimeter - Precise Color Analyzer Digital Color Reader with 8mm.

Nutrient digestibility

The effects of dietary treatments on the apparent digestibility coefficients of ash, Ca, and P at 35 days were different among treatments (P < 0.05, Table 6). Ash digestibility was the highest in the N30 and CP30 groups, and the lowest in the CONT, N15 and CP15 groups. Calcium digestibility was the highest in the CP15, N30 and CP30 groups and the lowest in the CONT group. The P digestibility was the highest in the N30 and CP30 groups and the lowest in the CONT group.

Table 6.

Effect of dietary treatments on the apparent digestibility coefficient at 35 days.

Parameters	CONT	N15	CP15	N30	CP30	SEM	P-value
Ash	59.86b	60.27b	63.95b	69.50a	71.01a	4.609	<0.01
Calcium	63.15b	65.10ab	70.04a	69.93a	72.52a	3.468	<0.01
Phosphorus	48.72c	51.25b	56.01ab	59.52a	63.48a	5.361	<0.01

^a-cvalues with different superscripts in the same row were significantly different (p < 0.05).

CONT: control diet formulated to meet Ca and aP requirements; N15: negative control diet reformulated to 15% lower Ca and aP compared CONT; CP15: negative control diet reformulated to 15% lower Ca and aP compared CONT supplemented with 300 mg/kg of calcium pidolate; N30: Negative control diet reformulated to 30% lower Ca and aP compared to the CONT; CP30: Negative control diet reformulated to 30% lower Ca and aP compared CONT supplemented with 300 mg/kg of calcium pidolate. SEM: Standard error of the mean (overall), n = 5 birds per treatment.

Litter characteristics

Litter analysis parameters (moisture, pH, Ca and P) at 35 days were not affected among dietary treatments (P > 0.05, Table 7). Litter moisture levels remained stable across all groups, ranging from 24.55% to 25.46%, with a pH ranging from 6.68 to 7.78. Similarly, Ca and P content in the litter showed minimal variation, with values ranging from 1.50 to 1.56% for Ca and from 0.87 to 0.91% for P.

Table 7.

Effect of dietary treatments on Litter analysis at age 35 days.

Parameters	CONT	N15	CP15	N30	CP30	SEM	P-value
Moisture	25.330	25.460	24.840	25.270	24.550	0.341	0.193
pH	6.990	6.680	7.410	7.780	7.600	0.403	0.069
Ca	1.530	1.510	1.560	1.510	1.500	0.021	0.271
P	0.910	0.880	0.900	0.890	0.870	0.014	0.067

CONT: control diet formulated to meet Ca and aP requirements; N15: negative control diet reformulated to 15% lower Ca and aP compared CONT; CP15: negative control diet reformulated to 15% lower Ca and aP compared CONT supplemented with 300 mg/kg of calcium pidolate; N30: Negative control diet reformulated to 30% lower Ca and aP compared to the CONT; CP30: Negative control diet reformulated to 30% lower Ca and aP compared CONT supplemented with 300 mg/kg of calcium pidolate. SEM: Standard error of the mean (overall).

Discussion

Optimizing the dietary Ca and P inclusion rate is of high interest in poultry diets, as Ca and P are critical for optimal production performance. Therefore, both excess and insufficient inclusion of these minerals might disrupt birds metabolism and production efficiency (Rousseau et al., 2016; Woyengo et al., 2022). This study aimed to evaluate the effect of including calcium pidolate in feeds with moderate (15%) and severe (30%) Ca and aP reductions on productive performance, blood parameters, bone quality, carcass traits, and nutrient digestibility in broilers.

The present study revealed that LBW and FCR are highly responsive to these dietary mineral levels. The inclusion of calcium pidolate in the 15% reduced diet resulted in the highest final LBW among treatments, with 3.5% and 8.5% higher LBW than CONT and N15, respectively. The CP15 group weight was also higher by 8.2% than the CP30 group. The best FCR value was achieved by the CP15 treatment, with similar levels in CONT and CP30 groups, and it was 8 points lower than the N15 group. Interestingly, a higher FI was observed for the CP15 group among the other treatments, with 90, 112, 146 and 176 g higher FI than the CONT, N15, N30 and CP30 groups, respectively. These results indicated that the moderate reduction of dietary Ca and aP (15% less than recommended levels), combined with a 300 mg/kg inclusion of calcium pidolate, effectively improves the production performance of broilers.

These results are in direct relation to the nutritional effect of calcium pidolate on broilers diets and its effect on the Ca and P metabolism. The decrease of Ca and aP by 15% in the N15 group negatively affected the production performance of the birds represented by a loss of LBW and feed conversion capacity. This is directly related to the importance of feeding Ca and aP according to the requirements (Shao et al., 2019; Li et al., 2020). Previous studies reported a similar effect when broilers were fed a deficient Ca and P diet, which negatively affected nutrient conversion efficiency and overall growth (Shao et al., 2019; Jabbar et al., 2024).

Maintaining Ca and P levels to the requirements is crucial due to their role in energy utilization and nutrient metabolism. Phosphorus is a key metabolite for several metabolic reactions to produce energy (Jabbar et al., 2024), and Ca plays a vital role in several metabolic processes, such as cell signaling, muscle contraction, enzyme function and fat deposition (Li et al., 2017).

Reducing the Ca and aP to 30% below the requirements drastically compromised the production capacity of the birds. For instance, the LBW of the N30 group was 2.4% lower than that of the N15 group, indicating that performance declines proportionally with increasing mineral restriction. Nonetheless, these results are not in agreement with previous studies. Valable et al. (2017) reported that feeding broilers 33% less of Ca and 23% less of aP didn’t affect the growth performance of broilers for LBW and FCR when broilers were fed a diet according to the NRC (1994) values. Additionally, bone mineral content and TBS value were affected by Ca and P deficiency, with a 14% and 18% decrease, respectively. Maintaining broiler performance is very important, but lower bone mineralization could strongly impair animal welfare due to skeletal disorders (Appelgate and Angel, 2008). In the present study, feeding requirements were according to FEDNA values. A slight difference is observed in Ca and aP values among the two feeding recommendations. For example, Ca requirements in the grower phase are 0.90 for NRC vs. 0.92% for FEDNA, and similar values for the aP. For the finisher phase, the Ca requirement is 0.85 for NRC vs. 0.80% for FEDNA, and for aP is 0.35% for NRC vs. 0.34% for FEDNA. We can observe that FEDNA values are lower than the NRC values; therefore, decreasing 30% of Ca and aP below recommendations resulted in a more aggressive challenge for the birds, which might explain the obtained results in the present study.

It is of note that the addition of 300 mg/kg of calcium pidolate was able to leverage this negative effect and production data of the CP30 group achieved similar performance to the CONT group, except for FI, which was lower for the control group. In accordance with this study, Fondevila et al. (2021) studied the effect of supplementing calcium pidolate at 300 mg/kg to broilers diet challenged with 3 levels of Ca and P reduction (10, 15 and 20% lower than the requirements). The authors reported that challenged birds tended to impair FCR value by 4 points at 21 days. However, calcium pidolate supplementation maintained the same FCR values through different Ca and P reduction as the control group at the same age (21 days). In contrast, ADG and ADFI were not affected by the type of treatment during the study. In similar results, Iglesias et al. (2023) reduced Ca and aP levels from 20 to 30% between pre-starter and starter phases. The authors reported that 300 mg/kg of calcium pidolate achieved similar results as the control group.

Production performance was different between the groups of calcium pidolate supplementation. The CP30 group had lower performance than the CP15 group. This result is important to understand the effect of calcium pidolate on broilers. It might be suggested that a higher decrease in the dietary Ca and aP should be accompanied by a higher inclusion rate of calcium pidolate in the diet to achieve the optimal production performance. However, further targeted investigations are required to validate this hypothesis and establish precise inclusion thresholds for varying degrees of mineral restriction. The inclusion of calcium pidolate without Ca and P challenge was also evaluated. Gül (2024) reported that the inclusion of calcium pidolate at 450 mg/kg on top of standard diets increased the production performance of broilers. Yet, a 600 mg/kg dose negatively affected broilers performance. In another study, Bertechini et al. (2024) reported that supplementing calcium pidolate from 1 to 21 days didn’t affect the production performance of broilers, but had an effect on decreasing birds pectoral myopathies.

Apparent digestibility coefficients for ash, Ca, and P were affected and were different among treatments. A higher digestibility value was observed for the reduced Ca and aP diets compared to the standard diet. The N30 group had 10% higher Ca digestibility and 22% P digestibility than the CONT, and the N15 had 3% higher Ca digestibility and 5% higher P digestibility. These results may indicate a compensatory mechanism to maximize mineral absorption when dietary levels are severely reduced (Gautier et al., 2017; Kim et al., 2017). Hoenderop et al. (2005) and Segawa et al. (2015) investigated this compensatory mechanism at the gene expression level in the intestine. The authors reported that Ca and P deficiency stimulated the expression of Ca transporters in the duodenum, like CALB1 and ATP2B1, and P transporters like SLC20A1 and SLC34A2.

This compensatory process helps to efficiently use the low existing levels of Ca and P from the diet. Similarly, Gautier et al. (2017) reported that Ca digestibility was increased by 20% in broilers fed Ca and P-deficient diets due to the adaptive mechanism. Interestingly, calcium pidolate supplementation further improved the digestibility coefficient for the birds. Calcium pidolate supplementation enhanced Ca and P digestibility for the CP15 and CP30 groups. The CP30 group exhibited a 30% increase in Ca digestibility and a 15% increase in P digestibility. Similarly, the CP15 group showed improvements of 11% for Ca and 15% for P compared to their respective controls. Even though a higher digestibility rate was observed with the CP30 group, it wasn’t enough to provide the required amount of Ca and P for optimal growth. Moreover, this result is also explained by the lower dietary intake in this challenged group (6% lower), where even a higher mineral digestibility wasn’t enough to maintain performance.

These data are important to show the role that calcium pidolate plays in enhancing Ca and P absorption and their assimilation by the bird. This effect is directly related to the effect of pidolic acid contribution to the Ca absorption mechanism in the intestinal cells through the activation of the calcium-binding protein that actively absorbs the Ca and drains it to the blood (Price, 2012; Bertechini et al., 2024).

Calcium pidolate is a calcium salt of pidolic acid. The interest in including calcium pidolate is because of its effect on calcium metabolism in birds. Calcium pidolate has been demonstrated to enhance calcium absorption and its mineralization in bones (Fondevila et al., 2021). This effect is attributed to pidolic acid because it is a precursor of arginine in the enterocytes (Price, 2012). Inside the intestinal cell, arginine enhances the expression of a calcium-binding protein, which is responsible for calcium active absorption from the gut (Price, 2012). In this way, calcium is actively absorbed in the gut and increases its availability in the blood. Additionally, pidolic acid is also a source of proline, which is a precursor of collagen formation in bones (Aguilon et al., 2024). Eventually, the increased availability of calcium in the blood will be efficiently mineralized in bones (Price, 2012; Aguilon et al., 2024).

Moreover, P absorption is also improved indirectly. The high absorption of Ca from the intestinal lumen decreases the contact of Ca with dietary phytate and avoids the formation of the calcium-phosphate precipitate or calcium-phytate complex (Krieg et al., 2021), which are indigestible and inaccessible for the phytase. This complex is a big concern in poultry nutrition, as it affects P liberation from phytate because the exogenous phytase cannot break down phytate in this case (David et al., 2023). Inversely, the decrease in the presence of this complex means higher phytase activity and higher P availability for the birds. This mechanism may explain the positive effect observed with calcium pidolate on mineral digestibility. However, despite higher mineral digestibility, the overall performance in the N30 group remained inferior, suggesting that increased nutrient absorption under severe restriction cannot fully counteract the negative impacts on growth and bone quality. Fondevila et al. (2021) study was in accordance with the herein data. The authors observed that supplementing reduced-mineral diets (15% Ca and P reduction) with calcium pidolate improved P digestibility by 12% at 14 days; conversely, Ca digestibility remained comparable to that of the control group. Furthermore, reducing dietary Ca contributes to a reduction in pH level because of the buffering capacity of calcium sources in the diet. As phytate availability increases with higher solubility at low pH, it results in a higher phytase activity and P liberation (Angel, 2019; Selle et al., 2023).

According to mineral digestibility, blood Ca and P were also affected by the type of treatments. Blood parameters serve as critical indicators of metabolic adaptation to dietary mineral availability. The N30 group exhibited the highest PTH and FGF23 levels, indicating a strong physiological response to Ca and P deficiency.

The PTH is secreted by the parathyroid gland and controls FGF23 secretion by osteocyte and osteoblast cells, and both are involved in Ca and P homeostasis (Ren et al., 2017; Agoro et al., 2020). The FGF23 hormone regulates P balance by decreasing its reabsorption in the kidney and its mineralization in bone along with Ca (Bergwitz and Juppner, 2010). The higher level of FGF23 is a sign of Ca and P imbalance in poultry, most likely due to higher dietary inclusion of these minerals (Gattineni et al., 2009). Higher FGF23 levels in the blood indicate the bird is regulating excess dietary P by increasing its excretion in urine and reducing its intestinal absorption (Gattineni et al., 2009).

Ren et al. (2017) reported that feeding laying hens with a deficient level of P had a lower blood level of FGF23, which is reasonable because of the compensatory effect to maintain P homeostasis in the blood. The higher level of FGF23 is a sign of Ca and P imbalance in poultry, most likely due to higher dietary inclusion of these minerals (Gattineni et al., 2009; Wang et al., 2018). Furthermore, while a reduction in dietary P typically corresponds with lower circulating FGF23 levels, the mineral-restricted groups in the present study (N30, N15, and CP30) unexpectedly exhibited elevated FGF23 levels. Consequently, lower P and Ca levels were observed in the blood in these treatments. Furthermore, P and Ca retention in tibia bone for N30 were also lower than those of the CONT group, explaining the relation between P and Ca availability and their retention in bones. This elevation in FGF23 concentrations contradicts the anticipated physiological response, as the birds adaptive mechanisms typically suppress FGF23 release under mineral restriction to maximize renal phosphorus retention. These results are in accordance with Wu et al. (2023). These authors also observed high FGF23 levels when broilers were fed low P and Ca diets. A high FGF23 level might lead to P metabolism alteration and substantially affect bone mineralization (Shimada et al., 2004). These results are intriguing and couldn’t be explained in basis of literature findings. A plausible explanation for these findings is that elevated FGF23 levels represent a compensatory response to intense bone mineral mobilization, triggered by dietary Ca and P deficiencies. This mechanism likely facilitates the liberation of excessive P into the bloodstream to maintain mineral balance. Given that FGF23 analyses were conducted at the end of the trial (35 days), it can be hypothesized that this adaptive response was initiated during the early life stages of the birds, contributing to the hormonal and mineral imbalances observed, a trend consistent with the findings of Meier et al. (2025). Further research is required to fully elucidate the dynamics of this physiological adaptation to restricted dietary Ca and P.

When calcium pidolate was supplemented in the CP30 group, lower levels of PTH and FGF23 were observed in the blood. This indicates that the birds suffered less from P and Ca deficiency. This result indicates that calcium pidolate improved P and Ca absorption from the intestine, and P and Ca blood levels were similar to the CONT group. Similarly, bone P content in the CP30 group was maintained at normal levels as in the CONT group; however, its Ca content was 12% lower. Decreasing Ca and P to 15% below requirements also activated the compensatory effect of the birds. The PTH was 25% higher and FGF23 was 19% higher in the blood. Consequently, lower Ca and P levels were observed in the blood than in the CONT group. Feeding calcium pidolate in the CP15 group showed similar levels of PTH and FGF23 as the CONT group, suggesting that calcium pidolate supplementation prevented excessive bone resorption and preserved mineral balance. It is also of note that calcium pidolate supplementation limited the activation of compensatory effects in the birds, by which, Ca and P content in bones was also maintained at normal levels.

These findings are consistent with prior research indicating that organic calcium compounds promote intestinal mineral absorption and reduce dependency on passive diffusion pathways (Fallah et al., 2018; Imari et al., 2022).

Along with PTH and FGF23 secretion to maintain Ca and P homeostasis, ALP also has an important role in this process. Alkaline phosphatase is an enzyme secreted by osteoblast cells to maintain calcium and P deposition in bones (Li et al., 2014). The secretion of ALP by osteoblast is stimulated by the rate of bone mineralization (Shao et al., 2019). Li et al. (2014) reported that ALP blood level was increased with a decrease of dietary P. Therefore, Ca and P deficiency impairs bone mineralization and development, which stimulates ALP secretion (Li et al., 2020).

In the present study, data of the Ca and P-deficient group (N30) exhibited 10% higher ALP blood concentration compared to the CONT group, which is in accordance with the study of (Li et al., 2020). Supplementing calcium pidolate to the deficient diet in the CP30 group wasn’t able to maintain lower levels of ALP compared to the N30 group. Even though calcium pidolate maintained blood Ca and P levels similar to those of the control group, it was not sufficient to sustain a lower ALP concentration. This finding confirms that ALP secretion is closely related to Ca and P deposition, as evidenced by the lower tibial Ca deposition observed in comparison with the control group. Additionally, calcium pidolate supplementation in the CP15 group maintained a similar level of ALP compared to the CONT group because it had similar Ca deposition as the CONT group as well.

Following the Ca and P digestibility response and blood availability, bone chemical analysis provides insight into mineral retention patterns and their subsequent utilization in supporting broiler growth and performance. Bone mineralization is strictly regulated by hormones PTH and FGF23, along with VD3 (Poorhemati et al., 2023; Reis et al., 2023). In general, Ca and P deficiency are expressed by bone resorption and osteoporosis (Li et al., 2020). Bone ash content is a critical measure of birds bone mineralization and is often lower in birds with bone disorders. Therefore, it is a widely used criterion to assess a poultry flock's skeletal health (Pritchard et al., 2020). Ash bone percentage reflects the inorganic, mineralized portion of the bone, and its deficiency leads to weaker and less mineralized bones, which are characteristic of bone disorders in poultry. The N30 and N15 groups had the lowest ash content among the treatments, indicating lower bone mineralization in birds fed a Ca- and P-deficient diet without calcium pidolate. Calcium content was also lower in the N30 and CP30 groups, followed by the N15 group. These results demonstrate high Ca mobilization from bones in response to dietary Ca and P deficits to maintain Ca homeostasis. Furthermore, since Ca serves as the primary substrate in the 1.67:1 hydroxyapatite ratio, its deficiency acts as the metabolic bottleneck for mineral deposition, regardless of P availability (Adji et al., 2025).

Notably, the CP15 group maintained bone ash and Ca levels comparable to the CONT group, indicating enhanced mineral deposition efficiency despite the dietary Ca and P deficiency. Similarly, the CP30 group presented higher P content than the N30 group and similar to the CP15 and CONT groups. This result indicates that calcium pidolate sustained P metabolism despite its dietary deficiency and limited P bone resorption.

The TBS followed a similar trend, with CP15 birds having 4 kg higher TBS compared to the N15 group and 1 kg higher than the CONT group. In accordance with this data, Rousseau et al. (2016) reported that when broilers were fed a Ca and P deficiency diet, tibia ash weight was reduced by 18% and ash content by 3.7%, and breaking strength by 21%. These findings suggest that 15% Ca and aP restriction combined with calcium pidolate supplementation is effective in preserving bone mineralization and strength, which indicates better Ca and P retention by the birds. In contrast, high restriction (N30) resulted in compromised skeletal integrity, which may predispose birds to bone disorders and reduced performance (Valable et al., 2017; Li et al., 2024). The combination of high dietary restriction with calcium pidolate supplementation in the CP30 group showed an intermediate value among the other treatments and a slight improvement in breaking strength (1.48 kg higher) than the N30 group. As bone strength and hardness are related to bone ash (Bonser and Casinos, 2003), TBS values followed the same trend as ash content percentage on tibia bone. This relation may explain the CP30 TBS intermediate value, even though it exhibited lower Ca content as the N30 group.

Morphometric bone traits (Length/Diameter) were less sensitive to dietary Ca and P reduction compared to bone mineral content. Birds bone traits are more often driven by genetics and age and are less sensitive to short-term mineral fluctuations than mineralization density is (Omotoso et al., 2023).

For carcass traits and muscle deposition, the breast muscle development is highly dependent on nutrient utilization efficiency, particularly Ca and P availability, which influence muscle protein synthesis and metabolic energy balance (Fatemi et al., 2021). The N30 and CP30 groups exhibited significantly lower breast yield compared to the control, suggesting that severe Ca and P restriction impairs protein deposition and muscle accretion independently of calcium pidolate supplementation. Reduced breast muscle yield under mineral-deficient conditions has been linked to metabolic energy redistribution, where the body prioritizes skeletal maintenance over muscle growth (Abdulla et al., 2017; Imari et al., 2022). It is also suggested that Ca and P requirements are higher for bone than for soft tissue, which unprioritized muscle growth in dietary reduced treatments (Xu et al., 2021). In contrast, the N15 and the CP15 groups maintained breast yield at the CONT level, indicating that 15% dietary Ca and aP restriction didn’t affect muscle growth.

Thigh and drumstick percentages were not significantly affected by dietary treatments, suggesting that Ca and P restrictions primarily impact muscle groups with higher protein turnover, such as the breast (pectoralis major). Although previous studies (Hakami et al., 2022; Bertechini et al., 2024) indicate that breast muscle development is highly sensitive to dietary mineral imbalances, further investigation is required to fully clarify the mechanisms underlying this relationship.

Lower dietary Ca and P is reported to decrease litter moisture and consequently footpad dermatitis in broilers (Rousseau et al., 2016). In the herein study, dietary Ca and P modification didn’t affect litter moisture retention or Ca and P levels among treatments. These results suggest that dietary Ca and P didn’t affect their environmental excretion, and the observed effects were more linked to their metabolism and efficiency of utilization by the animal.

In all, to the best of our knowledge, this is the first study to investigate the physiological and metabolic mechanisms of broiler chickens fed Ca and P-deficient diets supplemented with calcium pidolate. By integrating Ca and P digestibility data with blood mineral profiles and regulatory hormone analysis, this research provides a comprehensive understanding of how calcium pidolate enhances the overall growth performance of broiler chickens under mineral-restricted conditions.

These findings have practical implications for the poultry industry. By incorporating calcium pidolate into broiler diets formulated below standard Ca and aP levels, producers can potentially reduce feed costs while maintaining production efficiency and skeletal health. However, caution must be exercised to avoid excessive mineral restriction, as evidenced by the poor performance of birds in the N30 group, which experienced significant growth suppression, inefficient feed utilization, and compromised bone quality (Abdelhady et al., 2015; Rousseau et al., 2016; Imari et al., 2022)

Conclusion

This study confirmed that reducing Ca and P levels below the FEDNA recommendation levels affects broilers performance and bone mineralization. However, the study supports that the deleterious effects of the calcium and P reduced inclusion can be counteracted by the strategic use of calcium pidolate as a nutritional intervention to optimize mineral metabolism in broilers; this source of bioavailable calcium enhances performance and ensures skeletal integrity.

CRediT authorship contribution statement

Abdelhacib Kihal: Writing – review & editing, Supervision, Project administration, Methodology, Conceptualization. AbdelRahman Y. Abdelhady: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Salah A. El-Safty: Visualization, Supervision, Methodology, Formal analysis. Ahmed Radwan: Visualization, Supervision. Sergio Merinero: Writing – review & editing, Visualization, Software, Conceptualization. Monica Puyalto: Writing – review & editing, Resources, Conceptualization. Juan Jose Mallo: Writing – review & editing, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Conceptualization.

Disclosures

The authors declare no conflicts of interest. Although some authors are affiliated with Norel Animal Nutrition, Spain, this affiliation did not influence the experimental design, data collection, analysis, interpretation of results, or manuscript preparation. The study was conducted objectively, and the results reported herein accurately reflect the data obtained.