Effects of dietary calcium and phosphorus levels on broiler production and blood biochemistry in phytase-supplemented diets

Abstract

Numerous publications have reported the detrimental effects of calcium (Ca) over-feeding on broiler performance and nutrient digestibility, but its impact on blood biochemistry related to nutrient metabolism is not clear. The current study aimed to evaluate the effects of Ca and phosphorus (P) on broiler production and blood metabolites in phytase-supplemented diets. A 3 × 2 factorial arrangement was applied, including 3 Ca levels (Low, Medium or High) and 2 non-phytate P (nPP) levels (Marginal or Adequate). The experimental diets were corn-SBM based, and a phytase product (HiPhorius^TM, dsm-firmenich, Switzerland) at 1000 FYT/kg was supplied in all the diets. There were 40 birds/pen and 8 pens/treatment. The results showed significant interactions between Ca and P levels (P < 0.05) during the starter phase: High Ca increased (P < 0.05) feed conversion ratio (FCR) and plasma uric acid compared to Low Ca with Marginal nPP level, while High Ca improved (P < 0.05) tibia ash compared to Low Ca with Adequate nPP level. However, these interactions diminished as birds aged, only significant main effects of Ca level (P < 0.05) were observed on these parameters in the finisher phase. Compared to Low Ca, High Ca showed lower (P < 0.05) body weight (BW) gain and higher (P < 0.05) BW-corrected FCR at 0-42 d of age, higher (P < 0.05) tibia ash and breaking strength, and higher (P < 0.05) plasma uric acid at 43 or 44 d of age. The increase of plasma uric acid with High Ca may indicate a possible competition in the renal system to regulate nitrogen metabolism and Ca/P homeostasis, presumably compromising protein accretion. Additionally, High Ca showed significantly higher (P < 0.05) footpad lesion scores compared to Low Ca, especially with Marginal nPP level. In conclusion, a decrease of dietary Ca may improve growth performance and footpad lesion scores by alleviating renal burden through its hypocalciuretic effect, but this comes at the expense of reduced bone mineralization and breaking strength. Therefore, the dietary Ca level is recommended to be adjusted according to specific production objectives in broiler operations.

Introduction

Dietary calcium (Ca) and phosphorus (P) are essential for skeletal integrity and play critical roles in numerous metabolic processes (Underwood and Suttle, 1999). Inadequate supply of Ca and P can negatively impact bone development and growth performance (Shao et al., 2019). However, Ca oversupply rather than Ca deficiency is a common issue in commercial diets due to the low cost and abundant availability of limestone, the primary Ca source (David et al., 2023). Also, the Ca is usually not considered in feed formulation when limestone is used as flow agent in feed ingredients and diluting material in premixes, intensifying the issue of Ca oversupply (Sotak-Peper et al., 2016).

Numerous publications have reported the detrimental effects of Ca over-feeding on broiler performance (Powell et al., 2011; Amerah et al., 2014; Gautier et al., 2017; Krieg et al., 2021). This may be attributed to the formation of calcium-phytate complexes, which negatively affect the digestibility of P and amino acids (Tamim et al., 2004; Amerah et al., 2014; Mutucumarana et al., 2014). Additionally, excess dietary Ca was reported to deteriorate litter moisture and increased the incidence of footpad dermatitis (Rousseau et al., 2016). However, there are limited studies on the effects of high Ca on blood biochemical parameters related to nutrient metabolism.

It is known that the adverse effects of Ca over-feeding can be mitigated by phytase supplementation (Powell et al., 2011; Amerah et al., 2014; Krieg et al., 2021). Given the widespread use of phytase in poultry diets, it’s crucial to understand the impact of Ca on broiler performance in phytase-supplemented diets. Furthermore, the negative effects of high Ca are influenced by dietary P levels - particularly pronounced in low non-phytate P (nPP) diets (Rousseau et al., 2016; Gautier et al., 2017). This is likely due to the interdependent nutritional requirements of Ca and P, as increasing the concentration of one mineral (Ca or P) reduces the absorption efficiency of the other (Mutucumarana et al., 2014). Therefore, the objective of this study was to evaluate the interactions between dietary Ca and P on broiler production and blood biochemistry in phytase-supplemented diets, with Ca and P levels designed at commercially relevant concentrations.

Materials and methods

Birds, experimental design and diets

The animal protocol for this research was approved by the Animal Welfare Committee of dsm-firmenich (China) Animal Nutrition Research Center and complied with the guidelines in the European Union council directive 2010/63/EU for animal experiments. This experiment was conducted at dsm-firmenich Animal Nutrition Research Center Co., Ltd (Bazhou, China).

A total of 1,920 Cobb 500 male broilers were obtained on d of hatch and raised in floor pen covered with wood shavings. At arrival, birds were sorted by body weight (BW), transferred and randomly allocated to one of 6 dietary treatments. For each treatment, there were 8 pens with 40 birds per pen. The pen dimensions were 0.94 m × 0.8 m. All birds were reared in an environmentally controlled room with a lighting program of 23L: 1D during the first week and 20L: 4D afterward until the end of the trial. The temperature of the room was adjusted according to breed guidelines. Birds were allowed ad libitum access to feed and water. The trial lasted for 44 d comprising a 10-d starter, a 18-d grower and a 16-d finisher phase.

The 6 dietary treatments were in a 3 × 2 factorial arrangement including 3 Ca levels (Low, Medium or High) and 2 non-phytate P (nPP) levels (Marginal or Adequate). According to the range observed under commercial conditions, the Low, Medium or High Ca treatment was formulated to contain total Ca at 0.80, 0.95 or 1.10 % in the starter period, 0.70, 0.85 or 1.00 % in the grower period, and 0.55, 0.70 or 0.85 % in the finisher period, respectively. The Adequate or Marginal nPP treatment was formulated to contain nPP at 0.52 or 0.45 % in the starter period, 0.45 or 0.38 % in the grower period, and 0.41 or 0.34 % in the finisher period, respectively. To ensure graded concentrations of total Ca were achieved, a large batch of Low and High Ca diets were prepared and then combined at 50:50 ratio to create the Medium Ca diets. The diet composition and nutrient content of Low and High Ca diets were provided in Table 1. The experimental diets were corn-SBM based, and other nutrients were formulated to meet or exceed the bird’s requirements (Cobb, 2022). A novel phytase product (HiPhorius^TM, dsm-firmenich, Switzerland) with great improvement in intrinsic temperature and pH stability (Zhang et al., 2022) was supplied at 1000 FYT/kg in all the diets, contributing 0.16% total Ca and 0.19% nPP. All the diets were pelleted at 75-80 °C and supplied in crumbled form for the first week to ease intake.

Table 1.

Diet composition and nutrient content of the experimental diets, as-fed.

Ingredient (%)	Starter (d 0-10)				Grower (d 10-28)				Finisher (d 28-42)
	Low Ca, Adequate P	High Ca, Adequate P	Low Ca, Marginal P	High Ca, Marginal P	Low Ca, Adequate P	High Ca, Adequate P	Low Ca, Marginal P	High Ca, Marginal P	Low Ca, Adequate P	High Ca, Adequate P	Low Ca, Marginal P	High Ca, Marginal P
Corn	51.57	49.93	51.93	50.29	56.13	54.49	56.48	54.85	62.32	60.68	62.68	61.04
Soybean meal	40.84	41.14	40.78	41.08	36.75	37.05	36.68	36.98	31.37	31.67	31.30	31.60
Soya oil	4.17	4.69	4.06	4.58	4.19	4.71	4.08	4.59	3.98	4.50	3.87	4.39
Dicalcium phosphate	1.18	1.19	0.78	0.78	0.80	0.81	0.40	0.40	0.60	0.61	0.20	0.20
Limestone	0.55	1.37	0.78	1.60	0.52	1.34	0.75	1.57	0.26	1.08	0.49	1.31
Vit-Min Premix1	0.50	0.50	0.50	0.50	0.50	0.50	0.50	0.50	0.50	0.50	0.50	0.50
Salt	0.31	0.31	0.31	0.31	0.31	0.31	0.31	0.31	0.32	0.32	0.32	0.32
Sodium bicarbonate	0.24	0.23	0.24	0.23	0.24	0.23	0.24	0.23	0.23	0.22	0.23	0.22
DL-methionine	0.32	0.32	0.32	0.32	0.29	0.29	0.29	0.29	0.23	0.23	0.23	0.23
L-lysine HCl	0.14	0.13	0.14	0.13	0.13	0.12	0.13	0.13	0.09	0.09	0.10	0.09
L-threonine	0.11	0.11	0.11	0.11	0.08	0.08	0.08	0.08	0.03	0.03	0.03	0.03
Choline chloride	0.06	0.06	0.06	0.06	0.06	0.06	0.06	0.06	0.06	0.06	0.06	0.06
Phytase	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01
Total	100.00	100.00	100.00	100.00	100.00	100.00	100.00	100.00	100.00	100.00	100.00	100.00

Calculated energy and nutrient content
AME, kcal/kg	3000	3000	3000	3000	3050	3050	3050	3050	3100	3100	3100	3100
Crude protein, %	22.5	22.5	22.5	22.5	21.00	21.00	21.00	21.00	19.00	19.00	19.00	19.00
Digestible Lys, %	1.26	1.26	1.26	1.26	1.16	1.16	1.16	1.16	1.01	1.01	1.01	1.01
Digestible Met+Cys, %	0.94	0.94	0.94	0.94	0.88	0.88	0.88	0.88	0.78	0.78	0.78	0.78
Digestible Thr, %	0.86	0.86	0.86	0.86	0.78	0.78	0.78	0.78	0.66	0.66	0.66	0.66
Total Ca, %	0.80	1.10	0.80	1.10	0.70	1.00	0.70	1.00	0.55	0.85	0.55	0.85
Digestible Ca, %	0.35	0.49	0.35	0.49	0.30	0.44	0.31	0.45	0.23	0.37	0.24	0.38
Total P, %	0.75	0.75	0.69	0.68	0.68	0.68	0.61	0.61	0.63	0.63	0.56	0.56
Phytate P, %	0.23	0.23	0.24	0.23	0.23	0.23	0.23	0.23	0.22	0.22	0.22	0.22
non-phytate P, %	0.52	0.52	0.45	0.45	0.45	0.45	0.38	0.38	0.41	0.41	0.34	0.34
Digestible P, %	0.46	0.46	0.41	0.41	0.40	0.40	0.35	0.35	0.36	0.36	0.31	0.31

¹Vitamin-mineral premix provided (per kg of diet): Vitamin A 9800 IU, Vitamin D₃ 3500 IU, Vitamin E 45 IU, Vitamin K₃ 3 mg, Vitamin B₁ 3 mg, Vitamin B₂ 8 mg, Vitamin B₆ 4 mg, Vitamin B₁₂ 0.02 mg, Biotin 0.15 mg, Folic acid 2 mg, Niacinamide 60 mg, D-calcium pantothenate 15 mg, Fe 50 mg, Cu 15 mg, Mn 90 mg, Zn 70 mg, I 1 mg, Se 0.3 mg, Ca: 0.66 g.

Sampling and measurements

Feed consumption and BW by pen were recorded at d 0, 10, 28 and 42 of age for the calculations of weight gain (WG) and feed intake (FI). Mortality was recorded daily. The weight of dead birds was used to adjust the feed conversion ratio (FCR). Right tibia were collected from 4, 2 or 2 birds/pen at d 10, 28 or 44 of age, respectively. Right radius and ulna were collected from 2 birds/pen at d 44 of age. Tibia, radius and ulna were defleshed, and cartilaginous caps were removed after collection. They were kept frozen in plastic bags at -20 °C to maintain wetness until further analysis. Blood samples were collected from 1, 1 or 2 birds/pen at d 10, 28 or 43 of age, respectively, for the determination of blood biochemistry. Additionally, footpad dermatitis was assessed with 2 birds/pen at d 44, using a 4-point assessment scale adapted from the scoring scale in Welfare Quality® (2009). Specifically, scores 0, 1, and 2 in our assessment were consistent with the Welfare Quality® scale, while scores 3 and 4 were combined into a single score 3 in our assessment. Score 0 = no evidence of footpad lesion; score 1 = small black lesion; score 2 = moderate lesion; score 3 = severe lesion.

Chemical analyses

The samples of diet were ground to pass through a 0.5-mm screen before analysis. All samples were analyzed in duplicate. The samples were dried at 105°C in an oven for 4 h for dry matter determination (method 934.01; AOAC International, 2006). Nitrogen content was determined by the Dumas method (LECO FP-528, LECO Corporation, St Joseph, MI; method 992.23; AOAC International, 2006). Ca and P were determined by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES; Optima TM 8000, PerkinElmer, Shelton, CT; method 985.01; AOAC International, 2006) after sulfuric acid mineralization (Zhang et al., 2022). Phytase activity in diet samples was analyzed according to the method of Engelen et al. (1994). The fresh bone was determined for its breaking strength by a three-point bending test using a ​​TA.XT Plus texture analyzer (Stable Micro Systems, SMS)​​ equipped with a ​​HDP/3PB probe​​ (three-point bending fixture). The bone was ashed according to the method described by Kim et al. (2004) with modifications. Briefly, the bone samples were defatted with ethanol and ether, dried at 105 °C for 24 h to constant weight, ashed at 550 °C for 48 h and then analyzed Ca and P content.

Heparinized whole blood was analyzed for sodium, potassium, chloride, total carbon dioxide (CO₂), ionized Ca, anion gap, hematocrit, hemoglobin and pH using the i-STAT Alinity system (Abbott Laboratories, Chicago, IL) with i-STAT CHEM8+ cartridges (ref: ​​03P85​​, Abbott) according to manufacturer’s instructions. Aspartate aminotransferase (AST), creatine kinase (CK), uric acid (UA), glucose, total Ca, P, total protein, albumin and globulin were analyzed using the VetScan VS2 Analyzer (Zoetis Inc., Parsippany, NJ) with Avian/Reptilian Profile Plus cartridges (ref: ​​500-0041​​, Zoetis) according to manufacturer’s instructions.

Statistical analysis

Data were analyzed using JMP 16.0 (SAS Institute, Cary NC). Live performance, bone and blood data were analyzed by two-way ANOVA for Ca level, P level and their interaction; treatment means were separated by Fisher’s LSD test. The normality of data was checked, and log transformation was applied when needed. The treatment effect on footpad dermatitis was analyzed by Wilcoxon/Kruskal-Wallis test; data were expressed as score frequency, % and score mean values. Additionally, the correlations of footpad dermatitis with blood biomarkers at 43 d of age were analyzed by Spearman test. Pen was the experimental unit for growth performance, bone parameters, and blood biochemistry. Bird was the experimental unit for footpad dermatitis and its correlation with blood biochemistry at 43 d of age. The statistical significance was defined at P ≤ 0.05, and a tendency was considered at 0.05 < P ≤ 0.10.

Results

Chemical analyses of the diets

The analyzed chemical compositions and phytase activity of experimental diets were presented in Table 2. The analyzed total Ca values were in alignment with the targets. The analyzed crude protein and total P were marginally higher than the target values, but they were in good parallel among the treatments. The phytase activity was greater than the target, but within expected ranges when assay variation and product overages were considered.

Table 2.

Analyzed nutrient content and phytase activity in the experimental diets.

Code	Crude protein, %	Target total Ca1, %	Analyzed total Ca, %	Target total P1, %	Analyzed total P, %	Phytase recovery, FYT/kg
Starter (d 0-10)
Low Ca, Adequate P	23.11	0.64	0.64	0.56	0.60	1208
Mid Ca, Adequate P	23.04	0.79	0.81	0.56	0.62	1202
High Ca, Adequate P	23.39	0.94	0.98	0.56	0.62	1212
Low Ca, Marginal P	22.96	0.64	0.67	0.49	0.55	1267
Mid Ca, Marginal P	23.08	0.79	0.82	0.49	0.55	1353
High Ca, Marginal P	22.86	0.94	0.95	0.49	0.55	1199
Grower (d 10-28)
Low Ca, Adequate P	21.57	0.54	0.57	0.49	0.53	1292
Mid Ca, Adequate P	21.33	0.69	0.70	0.49	0.53	1080
High Ca, Adequate P	21.35	0.84	0.86	0.49	0.54	1211
Low Ca, Marginal P	21.76	0.54	0.57	0.42	0.46	1315
Mid Ca, Marginal P	22.08	0.69	0.71	0.42	0.48	1051
High Ca, Marginal P	21.90	0.84	0.85	0.42	0.47	1163
Finisher (d 28-42)
Low Ca, Adequate P	19.70	0.39	0.41	0.44	0.48	1303
Mid Ca, Adequate P	19.83	0.54	0.57	0.44	0.49	1311
High Ca, Adequate P	19.90	0.69	0.72	0.44	0.49	1345
Low Ca, Marginal P	19.96	0.39	0.42	0.37	0.41	1234
Mid Ca, Marginal P	20.38	0.54	0.58	0.37	0.42	1277
High Ca, Marginal P	20.09	0.69	0.72	0.37	0.42	1354

¹Target total Ca and total P were the values minus total Ca and nPP matrixes from phytase.

Growth performance

At the starter phase (0-10 d, Table 3), there was an interaction (P < 0.05) of Ca level and P level on FCR, that High Ca induced a higher (P < 0.05) FCR compared to Low Ca when nPP was marginally deficient, whereas no difference among the Ca treatments with Adequate nPP. Additionally, the main effects of Ca level and P level were significant (P < 0.001) for feed intake (FI) and weight gain (WG): High Ca and Marginal nPP treatments decreased (P < 0.05) FI and WG compared to their counterparts, respectively.

Table 3.

Effect of dietary calcium and phosphorus levels on growth performance of broilers from hatch to 42 d of age¹.

Ca level	P level	d 0-10			d 10-28			d 28-42			d 0-42
		FI (g/bird)	WG (g/bird)	FCR	FI (g/bird)	WG (g/bird)	FCR	FI (g/bird)	WG (g/bird)	FCR	FI (g/bird)	WG (g/bird)	FCR	BWcFCR
Low Ca	Adequate nPP	193	193	1.002 bc	1929 a	1501	1.285	2792	1725	1.621	4862	3417	1.423	1.423
Mid Ca	Adequate nPP	188	189	0.995 c	1926 a	1481	1.301	2788	1724	1.619	4846	3394	1.428	1.436
High Ca	Adequate nPP	184	184	0.997 c	1922 a	1471	1.302	2748	1704	1.614	4778	3340	1.431	1.457
Low Ca	Marginal nPP	185	185	0.999 bc	1922 a	1513	1.270	2749	1705	1.614	4811	3403	1.413	1.418
Mid Ca	Marginal nPP	185	184	1.008 ab	1855 b	1465	1.266	2804	1750	1.605	4787	3392	1.404	1.408
High Ca	Marginal nPP	178	176	1.012 a	1829 b	1417	1.291	2669	1660	1.609	4625	3253	1.422	1.477
	SEM	1.59	1.77	0.003	17.4	15.4	0.007	36.5	35.2	0.018	44.7	38.1	0.007	0.017
Main effect
Low Ca		189 a	189 a	1.001	1926	1507 a	1.278 b	2771 ab	1715	1.617	4836 a	3410 a	1.418	1.421 b
Mid Ca		186 a	186 a	1.002	1890	1473 b	1.283 b	2796 a	1737	1.612	4816 a	3393 a	1.416	1.422 b
High Ca		181 b	180 b	1.004	1875	1444 b	1.297 a	2708 b	1682	1.612	4702 b	3297 b	1.427	1.467 a
SEM		1.12	1.25	0.002	11.9	10.5	0.005	25.0	24.1	0.012	30.6	26.1	0.005	0.012
	Adequate nPP	188 a	189 a	0.998	1926	1485	1.296 a	2776	1718	1.618	4829 a	3384	1.428 a	1.439
	Marginal nPP	183 b	182 b	1.006	1869	1465	1.276 b	2741	1705	1.609	4741 b	3349	1.413 b	1.434
	SEM	0.90	1.00	0.002	9.6	8.5	0.004	20.2	19.4	0.010	24.7	21.0	0.004	0.010
P-value
Ca level		<0.001	<0.001	0.56	0.01	<0.001	0.01	0.04	0.25	0.94	0.006	0.006	0.24	0.01
P level		<0.001	<0.001	0.004	<0.001	0.12	<0.001	0.22	0.65	0.53	0.01	0.25	0.01	0.74
Interaction		0.33	0.67	0.02	0.04	0.11	0.15	0.38	0.55	0.96	0.42	0.45	0.43	0.35

¹Means in columns with no common superscript differ significantly (P < 0.05).

Ca = calcium, nPP = non-phytate phosphorus, FI = feed intake, WG = weight gain; FCR = feed conversion ratio, BWcFCR = body weight corrected feed conversion ratio.

At the grower phase (10-28 d, Table 3), there was an interaction (P < 0.05) of Ca level and P level on FI, that Medium and High Ca decreased (P < 0.05) FI compared to Low Ca when nPP was marginally deficient, whereas no difference among the Ca treatments with Adequate nPP. Additionally, the main effects showed a higher (P < 0.05) WG for Low Ca compared to its counterparts; a lower (P < 0.05) FCR for Low and Medium Ca compared to High Ca or Marginal nPP compared to Adequate nPP.

At the finisher phase (28-42 d, Table 3), the main effect of Ca level showed a higher (P < 0.05) FI for Medium Ca compared to High Ca, while there was no interaction or main effects on WG and FCR.

Additionally, the main effects of Ca and P levels were observed significant across the whole phase (0-42 d, Table 3). Lower (P < 0.05) FI, lower (P < 0.05) WG, and higher (P < 0.05) BW-corrected FCR were induced with High Ca treatment, when comparing to Low and Medium Ca treatments. Lower (P < 0.05) FI and lower (P < 0.05) FCR were observed with Marginal nPP treatment compared to Adequate nPP treatment.

Bone breaking strength, ash, Ca and P

At 10 d of age (Table 4), there was an interaction (P < 0.05) of Ca level and P level on tibia ash and tibia P, that High Ca with Adequate nPP treatment induced a higher (P < 0.05) tibia ash and P compared to Low Ca with Adequate nPP treatment, whereas no difference among the Ca treatments when nPP was marginally deficient. The main effect of P level was significant (P < 0.05) for tibia breaking strength (BS): birds fed Adequate nPP diet showed a higher value than that of Marginal nPP group.

Table 4.

Effect of dietary calcium and phosphorus levels on tibia parameters of broilers¹.

Ca level	P level	10 d				28 d				44 d
		Tibia BS (N)	Tibia ash (%)	Tibia Ca (%)	Tibia P (%)	Tibia BS (N)	Tibia ash (%)	Tibia Ca (%)	Tibia P (%)	Tibia BS (N)	Tibia ash (%)	Tibia Ca (%)	Tibia P (%)
Low Ca	Adequate nPP	62.00	46.98 b	15.71	8.31 bc	297.16	47.78	16.49	8.54	354.88	41.19	14.47	7.63
Mid Ca	Adequate nPP	65.47	47.69 a	16.04	8.44 ab	311.25	48.14	17.20	8.69	421.71	42.57	15.05	7.94
High Ca	Adequate nPP	67.14	48.18 a	16.17	8.49 a	302.21	47.88	16.93	8.59	452.18	43.70	15.46	8.14
Low Ca	Marginal nPP	61.08	46.85 b	15.98	8.27 bcd	304.89	47.51	16.83	8.63	393.31	41.75	14.65	7.70
Mid Ca	Marginal nPP	61.17	46.96 b	15.88	8.17 cd	293.66	47.98	16.69	8.60	417.72	42.71	15.22	7.96
High Ca	Marginal nPP	59.79	46.78 b	15.65	8.10 d	293.39	47.81	16.81	8.60	447.21	43.70	15.58	8.13
	SEM	2.24	0.20	0.26	0.06	10.28	0.41	0.23	0.11	19.92	0.57	0.21	0.11
Main effect
Low Ca		61.54	46.91	15.85	8.29	301.03	47.64	16.66	8.59	374.09 b	41.47 b	14.56 b	7.66 b
Mid Ca		63.32	47.32	15.96	8.31	302.45	48.06	16.95	8.65	419.71 a	42.64 a	15.13 a	7.95 a
High Ca		63.47	47.48	15.91	8.30	297.80	47.85	16.87	8.60	449.69 a	43.70 a	15.52 a	8.14 a
SEM		1.54	0.14	0.18	0.04	7.27	0.29	0.16	0.08	14.09	0.40	0.15	0.08
	Adequate nPP	64.87 a	47.62	15.97	8.41	303.54	47.94	16.88	8.61	409.59	42.49	14.99	7.90
	Marginal nPP	60.68 b	46.86	15.84	8.18	297.31	47.77	16.78	8.61	419.41	42.72	15.15	7.93
	SEM	1.27	0.12	0.15	0.04	5.93	0.24	0.13	0.07	11.50	0.33	0.12	0.06
P-value
Ca level		0.62	0.02	0.90	0.97	0.90	0.60	0.44	0.85	0.002	0.001	<0.001	<0.001
P level		0.02	<0.001	0.52	<0.001	0.46	0.62	0.60	0.96	0.55	0.62	0.38	0.75
Interaction		0.34	0.01	0.32	0.03	0.46	0.97	0.19	0.73	0.47	0.88	0.99	0.92

¹Means in columns with no common superscript differ significantly (P < 0.05).

Ca = calcium, nPP = non-phytate phosphorus, BS = breaking strength.

At 28 d of age (Table 4), there was no interaction or main effects on tibia BS, ash, Ca and P.

At 44 d of age, the tibia (Table 4) as well as the radius and ulna (Table 5) were collected for bone measurements. The main effect of Ca level for all these three bones showed higher (P < 0.05) bone BS, ash, Ca and P for birds fed Medium and High Ca diets compared to Low Ca diet, except that radius ash and P in Medium Ca group were not different from that of Low Ca group.

Table 5.

Effect of dietary calcium and phosphorus levels on radius and ulna parameters of broilers¹.

Ca level	P level	44 d				44 d
		Radius BS (N)	Radius ash (%)	Radius Ca (%)	Radius P (%)	Ulna BS (N)	Ulna ash (%)	Ulna Ca (%)	Ulna P (%)
Low Ca	Adequate nPP	88.89	40.64	14.50	7.25	169.63	36.61	12.60	6.60
Mid Ca	Adequate nPP	95.22	42.46	15.23	7.58	201.12	38.42	13.31	6.96
High Ca	Adequate nPP	95.36	42.90	15.25	7.59	215.06	39.10	13.55	7.07
Low Ca	Marginal nPP	87.32	40.77	14.48	7.21	181.43	37.35	12.91	6.74
Mid Ca	Marginal nPP	98.39	41.80	15.04	7.37	210.46	38.50	13.38	6.96
High Ca	Marginal nPP	97.99	44.08	15.73	7.79	227.83	40.16	13.98	7.25
	SEM	2.58	0.78	0.27	0.16	8.66	0.67	0.24	0.12
Main effect
Low Ca		88.11 b	40.71 b	14.49 b	7.23 b	175.53 b	36.98 b	12.75 b	6.67 b
Mid Ca		96.80 a	42.13 ab	15.13 a	7.47 ab	205.79 a	38.46 a	13.34 a	6.96 a
High Ca		96.67 a	43.49 a	15.49 a	7.69 a	221.44 a	39.63 a	13.76 a	7.16 a
SEM		1.77	0.55	0.19	0.12	6.12	0.48	0.17	0.09
	Adequate nPP	93.16	42.00	14.99	7.47	195.27	38.04	13.15	6.88
	Marginal nPP	94.56	42.21	15.08	7.46	206.57	38.67	13.42	6.99
	SEM	1.43	0.45	0.16	0.09	5.00	0.39	0.14	0.07
P-value
Ca level		0.001	0.004	0.003	0.03	<0.001	0.001	<0.001	0.001
P level		0.48	0.74	0.69	0.91	0.12	0.26	0.18	0.29
Interaction		0.57	0.50	0.45	0.46	0.98	0.76	0.76	0.75

¹Means in columns with no common superscript differ significantly (P < 0.05).

Ca = calcium, nPP = non-phytate phosphorus, BS = breaking strength.

Blood biochemistry

At 10 d of age (Table 6a, Table 6b), there was an interaction (P < 0.05) of Ca level and P level on plasma uric acid, that a higher uric acid concentration was induced by High Ca treatment compared to Low Ca treatment when nPP was marginally deficient, whereas no difference among the Ca treatments with Adequate nPP. Also, the High Ca treatment showed a higher (P < 0.05) plasma Ca concentration and lower (P < 0.05) plasma P concentration compared to Low Ca treatment. The Adequate nPP treatment demonstrated higher (P < 0.05) concentrations of plasma P and glucose.

Table 6a.

Effect of dietary calcium and phosphorus levels on blood biochemistry of broilers at 10 d of age¹.

Ca level	P level	Sodium (mmol/L)	Potassium (mmol/L)	Chloride (mmol/L)	Total CO2 (mmol/L)	Ionized Ca (mmol/L)	Anion gap (mmol/L)	Hematocrit (%PCV)	Hemoglobin (g/dL)	pH
Low Ca	Adequate nPP	139.88	4.34	104.50	19.00	1.13	21.63	17.88	6.08	7.60
Mid Ca	Adequate nPP	137.63	4.60	102.13	21.13	1.09	19.88	18.67	6.35	7.60
High Ca	Adequate nPP	139.29	4.41	103.75	21.63	1.23	18.50	17.50	5.95	7.61
Low Ca	Marginal nPP	136.75	5.15	102.13	21.00	1.14	19.50	16.86	5.73	7.64
Mid Ca	Marginal nPP	138.63	3.89	103.00	19.00	1.02	21.75	18.17	6.17	7.61
High Ca	Marginal nPP	138.00	4.15	102.63	20.88	1.15	19.75	18.50	6.30	7.62
	SEM	1.31	0.36	1.43	1.21	0.07	1.18	0.74	0.25	0.03
Main effect
Low Ca		138.31	4.74	103.31	20.00	1.14	20.56	17.37	5.90	7.62
Mid Ca		138.13	4.24	102.56	20.06	1.05	20.81	18.42	6.26	7.60
High Ca		138.64	4.28	103.19	21.25	1.19	19.13	18.00	6.13	7.62
SEM		0.90	0.24	1.01	0.86	0.04	0.83	0.52	0.18	0.02
	Adequate nPP	138.93	4.45	103.46	20.58	1.15	20.00	18.01	6.13	7.60
	Marginal nPP	137.79	4.40	102.58	20.29	1.10	20.33	17.84	6.07	7.62
	SEM	0.72	0.20	0.83	0.70	0.04	0.68	0.40	0.14	0.01
P-value
Ca level		0.92	0.25	0.86	0.51	0.11	0.31	0.33	0.33	0.81
P level		0.27	0.81	0.46	0.77	0.38	0.73	0.76	0.76	0.36
Interaction		0.25	0.11	0.53	0.23	0.75	0.20	0.29	0.28	0.83

¹Means in columns with no common superscript differ significantly (P < 0.05).

Ca = calcium, nPP = non-phytate phosphorus, CO₂ = carbon dioxide.

Table 6b.

Effect of dietary calcium and phosphorus levels on blood biochemistry of broilers at 10 d of age¹.

Ca level	P level	AST (U/L)	CK (U/L)	UA (mg/dL)	Glucose (mg/dL)	Total Ca (mg/dL)	P (mg/dL)	Total protein (g/dL)	Albumin (g/dL)	Globulin (g/dL)
Low Ca	Adequate nPP	162.71	1961.71	7.65 b	259.38	11.41	8.00	2.53	1.60	0.96
Mid Ca	Adequate nPP	176.86	1817.13	9.09 ab	278.75	11.66	7.19	2.50	1.55	0.96
High Ca	Adequate nPP	183.00	1907.14	7.51 b	258.00	11.88	7.40	2.38	1.53	0.89
Low Ca	Marginal nPP	176.29	1767.63	7.58 b	254.75	11.53	7.31	2.55	1.63	0.97
Mid Ca	Marginal nPP	163.00	1422.00	7.86 ab	235.88	11.73	6.21	2.44	1.44	0.99
High Ca	Marginal nPP	188.00	1718.25	9.66 a	235.88	12.63	6.48	2.61	1.63	0.95
	SEM	11.69	233.59	0.69	12.00	0.24	0.36	0.07	0.06	0.08
Main effect
Low Ca		169.50	1864.67	7.61	257.06	11.47 b	7.66 a	2.54	1.61	0.97
Mid Ca		169.93	1619.56	8.48	257.31	11.69 b	6.70 b	2.47	1.49	0.98
High Ca		185.50	1812.70	8.58	246.94	12.25 a	6.94 b	2.49	1.58	0.92
SEM		8.27	159.93	0.47	8.49	0.17	0.25	0.05	0.04	0.05
	Adequate nPP	174.19	1895.33	8.08	265.38 a	11.65	7.53 a	2.47	1.56	0.94
	Marginal nPP	175.76	1635.96	8.36	242.17 b	11.96	6.67 b	2.53	1.56	0.97
	SEM	6.75	132.02	0.38	6.93	0.14	0.21	0.04	0.03	0.04
P-value
Ca level		0.25	0.62	0.27	0.61	0.008	0.03	0.66	0.13	0.69
P level		0.98	0.14	0.60	0.02	0.14	0.005	0.31	0.93	0.66
Interaction		0.49	0.83	0.04	0.29	0.32	0.91	0.11	0.19	0.93

¹Means in columns with no common superscript differ significantly (P < 0.05).

Ca = calcium, nPP = non-phytate phosphorus, AST = aspartate aminotransferase, CK = creatine kinase, UA = uric acid.

At 28 d of age (Table 7a, Table 7b), a higher (P < 0.05) plasma P concentration was observed with Adequate nPP treatment vs Marginal nPP treatment, while no treatment effect was detected for other plasma parameters.

Table 7a.

Effect of dietary calcium and phosphorus levels on blood biochemistry of broilers at 28 d of age¹.

Ca level	P level	Sodium (mmol/L)	Potassium (mmol/L)	Chloride (mmol/L)	Total CO2 (mmol/L)	Ionized Ca (mmol/L)	Anion gap (mmol/L)	Hematocrit (%PCV)	Hemoglobin (g/dL)	pH
Low Ca	Adequate nPP	144.63	4.96	109.25	23.75	1.35	17.75	22.88	7.80	7.47
Mid Ca	Adequate nPP	144.25	4.46	108.63	22.88	1.38	18.00	23.25	7.90	7.48
High Ca	Adequate nPP	144.43	4.85	108.57	23.88	1.31	17.86	22.00	7.49	7.47
Low Ca	Marginal nPP	143.75	4.71	108.13	25.00	1.32	17.14	22.38	7.59	7.49
Mid Ca	Marginal nPP	144.25	4.53	106.88	25.25	1.37	17.38	23.86	8.11	7.49
High Ca	Marginal nPP	144.63	4.54	109.75	23.50	1.39	17.43	23.50	8.00	7.48
	SEM	0.80	0.30	1.11	0.89	0.04	0.82	0.77	0.26	0.02
Main effect
Low Ca		144.19	4.84	108.69	24.38	1.34	17.45	22.63	7.69	7.48
Mid Ca		144.25	4.49	107.75	24.06	1.37	17.69	23.55	8.01	7.48
High Ca		144.53	4.69	109.16	23.69	1.35	17.64	22.75	7.74	7.47
SEM		0.55	0.21	0.76	0.61	0.03	0.58	0.53	0.18	0.02
	Adequate nPP	144.43	4.76	108.82	23.50	1.35	17.87	22.71	7.73	7.47
	Marginal nPP	144.21	4.59	108.25	24.58	1.36	17.32	23.24	7.90	7.48
	SEM	0.44	0.17	0.61	0.49	0.02	0.47	0.43	0.14	0.01
P-value
Ca level		0.89	0.52	0.40	0.73	0.57	0.95	0.40	0.41	0.93
P level		0.72	0.50	0.51	0.13	0.69	0.40	0.39	0.42	0.53
Interaction		0.75	0.80	0.36	0.23	0.28	0.99	0.34	0.30	0.89

¹Means in columns with no common superscript differ significantly (P < 0.05).

Ca = calcium, nPP = non-phytate phosphorus, CO₂ = carbon dioxide.

Table 7b.

Effect of dietary calcium and phosphorus levels on blood biochemistry of broilers at 28 d of age¹.

Ca level	P level	AST (U/L)	UA (mg/dL)	Glucose (mg/dL)	Total Ca (mg/dL)	P (mg/dL)	Total protein (g/dL)	Albumin (g/dL)	Globulin (g/dL)
Low Ca	Adequate nPP	306.00	4.15	257.43	11.11	7.96	3.05	2.10	0.94
Mid Ca	Adequate nPP	236.75	3.86	253.50	11.45	7.71	3.29	2.20	1.09
High Ca	Adequate nPP	237.75	5.00	258.13	11.06	7.76	3.25	2.21	1.03
Low Ca	Marginal nPP	279.00	4.48	257.88	11.09	7.28	3.23	2.25	0.98
Mid Ca	Marginal nPP	270.63	4.10	258.88	11.29	7.34	3.13	2.19	1.09
High Ca	Marginal nPP	275.63	4.30	259.50	11.50	6.98	3.20	2.24	1.10
	SEM	23.99	0.48	5.60	0.22	0.18	0.09	0.07	0.07
Main effect
Low Ca		292.50	4.31	257.65	11.10	7.62	3.14	2.18	0.96
Mid Ca		253.69	3.98	256.19	11.37	7.53	3.21	2.19	1.09
High Ca		256.69	4.65	258.81	11.28	7.37	3.23	2.23	1.06
SEM		16.96	0.34	3.84	0.16	0.13	0.06	0.05	0.05
	Adequate nPP	260.17	4.34	256.35	11.21	7.81 a	3.20	2.17	1.02
	Marginal nPP	275.08	4.29	258.75	11.29	7.20 b	3.18	2.23	1.05
	SEM	13.85	0.28	3.10	0.13	0.10	0.05	0.04	0.04
P-value
Ca level		0.18	0.41	0.88	0.47	0.38	0.57	0.76	0.11
P level		0.52	0.89	0.58	0.65	<0.001	0.88	0.37	0.48
Interaction		0.17	0.51	0.89	0.38	0.49	0.16	0.48	0.85

¹Means in columns with no common superscript differ significantly (P < 0.05).

Ca = calcium, nPP = non-phytate phosphorus, AST = aspartate aminotransferase, CK = creatine kinase, UA = uric acid.

At 43 d of age (Table 8a, Table 8b), higher (P < 0.05) concentrations of plasma uric acid and globulin were observed for High Ca compared to Low Ca or for Marginal nPP compared to Adequate nPP. Additionally, a higher (P < 0.05) plasma total protein was observed for High Ca compared to Low Ca.

Table 8a.

Effect of dietary calcium and phosphorus levels on blood biochemistry of broilers at 43 d of age¹.

Ca level	P level	Sodium (mmol/L)	Potassium (mmol/L)	Chloride (mmol/L)	Total CO2 (mmol/L)	Ionized Ca (mmol/L)	Anion gap (mmol/L)	Hematocrit (%PCV)	Hemoglobin (g/dL)	pH
Low Ca	Adequate nPP	147.19	4.78	109.38	25.50	1.39	17.69	24.38	8.29	7.43
Mid Ca	Adequate nPP	149.50	4.91	111.19	26.13	1.39	18.06	23.75	8.07	7.41
High Ca	Adequate nPP	147.69	4.78	110.44	26.06	1.38	17.44	24.06	8.18	7.43
Low Ca	Marginal nPP	147.19	4.66	109.56	25.81	1.37	17.56	24.38	8.29	7.45
Mid Ca	Marginal nPP	148.56	4.84	110.81	25.63	1.45	18.50	24.57	8.36	7.41
High Ca	Marginal nPP	149.06	4.74	111.44	25.94	1.41	17.63	25.13	8.54	7.40
	SEM	0.87	0.19	0.91	0.64	0.02	0.56	0.59	0.20	0.01
Main effect
Low Ca		147.19	4.72	109.47	25.66	1.38	17.63	24.38	8.29	7.44
Mid Ca		149.03	4.88	111.00	25.88	1.42	18.28	24.16	8.21	7.41
High Ca		148.38	4.76	110.94	26.00	1.39	17.53	24.59	8.36	7.41
SEM		0.62	0.13	0.64	0.45	0.01	0.40	0.40	0.14	0.01
	Adequate nPP	148.13	4.82	110.33	25.90	1.39	17.73	24.06	8.18	7.42
	Marginal nPP	148.27	4.75	110.60	25.79	1.41	17.90	24.69	8.39	7.42
	SEM	0.50	0.11	0.53	0.37	0.01	0.33	0.32	0.11	0.01
P-value
Ca level		0.11	0.69	0.18	0.87	0.20	0.36	0.72	0.72	0.15
P level		0.84	0.62	0.72	0.90	0.16	0.72	0.18	0.17	0.73
Interaction		0.42	0.97	0.75	0.83	0.11	0.88	0.61	0.61	0.37

¹Means in columns with no common superscript differ significantly (P < 0.05).

Ca = calcium, nPP = non-phytate phosphorus, CO₂ = carbon dioxide.

Table 8b.

Effect of dietary calcium and phosphorus levels on blood biochemistry of broilers at 43 d of age¹.

Ca level	P level	AST (U/L)	UA (mg/dL)	Glucose (mg/dL)	Total Ca (mg/dL)	P (mg/dL)	Total protein (g/dL)	Albumin (g/dL)	Globulin (g/dL)
Low Ca	Adequate nPP	488.69	2.95	238.31	11.44	7.44	3.41	2.31	1.11
Mid Ca	Adequate nPP	585.69	3.31	233.38	11.46	7.33	3.44	2.31	1.13
High Ca	Adequate nPP	536.56	3.34	241.50	11.57	7.28	3.46	2.30	1.16
Low Ca	Marginal nPP	466.13	3.31	243.69	11.38	7.19	3.35	2.23	1.11
Mid Ca	Marginal nPP	552.31	4.02	242.50	11.88	7.23	3.45	2.28	1.23
High Ca	Marginal nPP	474.75	4.74	236.75	11.82	7.10	3.68	2.36	1.36
	SEM	46.15	0.35	4.92	0.15	0.23	0.08	0.05	0.06
Main effect
Low Ca		477.41	3.13 b	241.00	11.41	7.32	3.38 b	2.27	1.11 b
Mid Ca		569.00	3.66 ab	237.94	11.67	7.28	3.44 ab	2.30	1.18 ab
High Ca		505.66	4.04 a	239.13	11.69	7.19	3.57 a	2.33	1.26 a
SEM		32.63	0.25	3.48	0.11	0.17	0.05	0.04	0.04
	Adequate nPP	536.98	3.20 b	237.73	11.49	7.35	3.44	2.31	1.13 b
	Marginal nPP	497.73	4.02 a	240.98	11.69	7.17	3.49	2.29	1.23 a
	SEM	26.64	0.20	2.84	0.09	0.13	0.04	0.03	0.03
P-value
Ca level		0.14	0.03	0.82	0.13	0.86	0.03	0.58	0.03
P level		0.42	0.01	0.42	0.11	0.36	0.34	0.67	0.04
Interaction		0.77	0.49	0.35	0.28	0.94	0.14	0.46	0.17

¹Means in columns with no common superscript differ significantly (P < 0.05).

Ca = calcium, nPP = non-phytate phosphorus, AST = aspartate aminotransferase, CK = creatine kinase, UA = uric acid.

Footpad dermatitis

At 44 d of age, High Ca treatment induced a significantly worse (P < 0.05) footpad dermatitis compared to Low Ca treatment when nPP was adequately supplemented, whereas both High and Medium Ca treatments showed significantly higher (P < 0.05) footpad lesion scores compared to Low Ca treatment when nPP was marginally supplemented (Table 9).

Table 9.

Effect of dietary calcium and phosphorus levels on footpad dermatitis of broilers at 44 d of age¹.

Ca level	P level	Footpad dermatitis
		Score2 frequency				Score mean
		0	1	2	3
Low Ca	Adequate nPP	50.0 %	43.8 %	6.3 %	0.0 %	0.56 bc
Mid Ca	Adequate nPP	31.3 %	50.0 %	18.8 %	0.0 %	0.88 ab
High Ca	Adequate nPP	18.8 %	56.3 %	25.0 %	0.0 %	1.06 a
Low Ca	Marginal nPP	56.3 %	43.8 %	0.0 %	0.0 %	0.44 c
Mid Ca	Marginal nPP	12.5 %	56.3 %	31.3 %	0.0 %	1.19 a
High Ca	Marginal nPP	6.3 %	56.3 %	25.0 %	12.5 %	1.44 a
	P-value	0.001
Low Ca		53.1 %	43.8 %	3.1 %	0.0 %	0.50
Mid Ca		21.9 %	53.1 %	25.0 %	0.0 %	1.03
High Ca		12.5 %	56.3 %	25.0 %	6.3 %	1.25
P-value		<0.001
	Adequate nPP	33.3 %	50.0 %	16.7 %	0.0 %	0.83
	Marginal nPP	25.0 %	52.1 %	18.8 %	4.2 %	1.02
	P-value	0.28

¹Means in columns with no common superscript differ significantly (P < 0.05).

²Footpad dermatitis: 0, no evidence of footpad lesion; 1, small black lesion; 2, moderate lesion; 3, severe lesion.

Ca = calcium, nPP = non-phytate phosphorus.

The footpad dermatitis was scored from the same birds used for blood biochemistry at 43 d of age. Therefore, the correlations of footpad dermatitis with selected blood biomarkers were performed and presented at Table 10. The results showed that footpad lesion scores didn’t correlate with plasma Ca and P, but it had a tendency to correlate with plasma uric acid (ρ = 0.17, P = 0.10), and significantly correlated with plasma globulin (ρ = 0.21, P < 0.05).

Table 10.

Correlations of footpad dermatitis with blood biomarkers of broilers at 43 d of age.

Blood biomarker	Correlation with footpad dermatitis
	ρ	P-value
Ca, mg/dL	0.09	0.39
P, mg/dL	-0.11	0.30
UA, mg/dL	0.17	0.10
Globulin, g/dL	0.21	0.04

Ca = calcium, P = phosphorus, UA = uric acid.

Discussion

Previous studies have reported that phytase supplementation could alleviate the negative consequences of excessive dietary Ca (Powell et al., 2011; Amerah et al., 2014; Krieg et al., 2021). This effect is primarily due to the hydrolysis of phytate by phytase, which increases the level of available P and consequently modulates the Ca to P ratio. The inclusion of phytase in the basal with its Ca and P matrix values enabled the investigation of Ca and P interactions under conditions relevant to commercial practices. The results of the current study revealed that the influence of dietary Ca and P levels on broiler performance varied across different growth phases. In the starter phase, high Ca levels significantly worsened feed efficiency when nPP was marginally deficient. Also, both high Ca and marginal nPP deficiency suppressed feed intake and body weight gain. These results suggested a narrow tolerance to inappropriate Ca and P levels and their imbalances in young chicks (Bar et al., 2003; Zhang et al., 2024). Gautier et al. (2017) demonstrated that the Ca to P ratio appeared to be more critical than the absolute concentrations of either mineral. Their findings revealed that growth performance was significantly reduced when increasing dietary Ca with unchanged nPP concentration (widening Ca to nPP ratio). Whereas the growth response remained unaffected when decreasing both minerals proportionally to maintain their ratio. The aforementioned sensitivity diminished as birds aged. In the grower phase, Ca and nPP imbalances still affected feed intake, but the magnitude of growth depression was attenuated compared to earlier observations. In the finisher phase, only minor effect on feed intake was detected with high Ca diets. Rousseau et al. (2016) reported the adaptive capacity of older birds may be attributed to the modulation of intestinal Ca and P transporters and consequently the digestive efficiency when facing dietary changes in Ca and P.

Similarly, bone quality responses to dietary Ca and P levels differed among growth stages. During the early growth phase (10 d of age), high Ca improved tibia mineralization only when combined with adequate nPP, indicating sufficient Ca and P were crucial for optimal bone development. Also, this early period represents a critical window where adequate P serves dual purposes: supporting skeletal development while preventing the growth depression with high Ca supplementation (Bar et al., 2003; Bayley et al., 2024). By market age (44 d of age), the persistent low Ca provision resulted in compromised bone mineralization and breaking strength across all measured bones (tibia, radius, and ulna), whereas it was noticed that low Ca maintained growth performance. These findings aligned with previous reports that Ca requirements for optimal growth were lower than those needed for skeletal development (David et al., 2023; Walk et al., 2024). Notably, the compromised bone strength carries important economic implications due to its negative impacts on processing yields and carcass quality. For example, bone fragility increases the risk of bone fragments in meat products and causes adjacent meat discoloration from blood leaching, which may reduce product appeal to consumers (Rath et al., 2000). These effects are particularly significant for radius and ulna bones, whose structural integrity directly determines the value of wing portions.

The analysis of plasma biochemical parameters provided important insights into the effects of dietary Ca and P levels on broiler growth and development. It’s known that plasma Ca and P concentrations are tightly regulated through hormones that coordinate intestinal absorption, renal excretion, and bone mobilization. These regulatory mechanisms maintain an inverse relationship between circulating Ca and P levels, so that a rise in plasma Ca typically accompanies a fall in P, and vice versa. This inverse relationship reflects their interdependent homeostatic control (Shafey, 1993; Proszkowiec-Weglarz and Angel, 2013). In the current study, high Ca supply induced an increase of plasma Ca (total) and a decrease of plasma P at 10 d of age. Marginal nPP deficiency induced lower plasma P at both 10 d and 28 d of age, but it had no influence on plasma Ca. These results were consistent with the previous observations by Cowesion et al. (2024). Specifically, the changes of Ca supply impacted both plasma Ca and P, with effect on P being more pronounced and consistent. Conversely, the changes of P supply primarily influenced plasma P rather than Ca, and this effect tended to persist longer. Interestingly, an increase of plasma uric acid was observed under high Ca with marginal P at 10 d of age. This may reflect the potential competition in the renal system to regulate nitrogen metabolism and Ca/P homeostasis, considering the reported calciuretic effects of protein intake (Linkswiler et al., 1981; Cowieson et al., 2020). The adequate supply of P appeared to offset the renal competition in the current study. It was probably by enhancing Ca retention to avoid Ca:P imbalance (Proszkowiec-Weglarz and Angel, 2013) and also perhaps through provision of P for ATP synthesis, thereby reducing protein catabolism and promoting protein accretion (Cowieson et al., 2020). The higher plasma glucose with adequate P at 10 d of age may further provide the metabolic fuel to promote growth performance through its roles in ATP generation and protein synthesis (Kwakye et al., 2025).

The attenuation of plasma Ca and P fluctuations over time probably suggested the maturation of Ca/P homeostatic mechanisms, which may explain the observed reduction in sensitivity to dietary Ca/P imbalance with older birds. However, the elevation of plasma uric acid and globulin at 43 d of age suggested long-term metabolic consequences of diet supplementation with high Ca or marginal nPP. An increase of plasma total protein was also detected with high Ca at 43 d of age. This increase was primarily driven by the rise of plasma globulin, as plasma albumin remained unaffected by Ca level. The sustained high plasma uric acid may reflect continued protein catabolism and enduring renal metabolic stress. The increase of plasma globulin may indicate chronic stress or inflammation of birds (Rezende et al., 2017), which was typically observed in birds with lower feed efficiency (Adeleye et al., 2018; Livingston et al., 2020). These findings demonstrated the metabolic costs of Ca/P imbalance persisted throughout the production cycle, although older birds can maintain plasma Ca/P homeostasis. Furthermore, the current study confirmed that plasma biomarkers can serve as valuable indicators for both nutritional metabolism and growth performance in broilers as suggested by Livingston et al. (2020).

The detrimental effect of high Ca on footpad dermatitis may be linked to poor litter quality, particularly via increased litter moisture from elevated urinary Ca excretion (Collett, 2012; Rousseau, 2016). As discussed earlier, the increased Ca excretion through urine may represent part of the Ca/P homeostasis mechanism in older birds to maintain blood Ca and P levels. This presumably explains the lack of direct correlation between footpad lesions and blood Ca/P levels at 43 d of age. Notably, footpad lesions showed a tendency to correlate with plasma uric acid in the current study. The increased plasma uric acid has been shown to be associated with higher excreted uric acid (Namroud et al., 2008). It may provide substrate for ammonia-producing bacteria that result in footpad irritation and eventually develop to dermatitis (Meluzzi et al., 2008). Furthermore, the positive correlation between footpad lesions and plasma globulin may reflect chronic stress and systemic inflammation of birds resulting from the dermatitis. Our results demonstrated that increasing nPP level from marginal to adequate improved the footpad lesion scores, which was potentially attributed to its role in mitigating renal competition as previously discussed (Proszkowiec-Weglarz and Angel, 2013; Cowieson et al., 2020).

In conclusion, the current study provided compelling evidence that blood biochemical parameters could serve as sensitive indicators of broiler metabolic responses to dietary Ca and P levels. The plasma mineral profiles (Ca and P concentrations) and metabolic markers (uric acid and globulin) revealed distinct homeostatic challenges at different growth stages, which explained the differences in growth performance and bone development across phases. During the starter phase, high Ca diets (particularly with marginal nPP) resulted in an increase of plasma Ca but a decrease of P, and also an elevation of uric acid. These changes reflected significant metabolic stress in renal function regarding protein metabolism and Ca/P homeostasis, which may account for the impaired growth and bone mineralization. The maturation of mineral homeostasis mechanisms was evident through the stabilization of plasma Ca and P levels in older birds. However, the persistent elevation of uric acid and globulin at market age revealed ongoing metabolic costs of high Ca diets. Collectively, our findings revealed a nutritional trade-off in broiler production. A decrease of dietary Ca improved growth performance and footpad lesion scores, probably by alleviating renal burden through its hypocalciuretic effect. However, this benefit came at the expense of impaired bone mineralization and skeletal strength. Therefore, the dietary Ca level is recommended to be tailored based on specific production goals. A thorough evaluation of dietary Ca, P, and phytase on protein metabolism is also suggested to optimize sustainability of broiler production.

CRediT authorship contribution statement

Qian Zhang: Conceptualization, Formal analysis, Writing – original draft. Hui Zhang: Investigation. Jingcheng Zhang: Investigation. Shikui Wang: Investigation. Zhenzhen Wang: Investigation. Stephane Duval: Writing – review & editing. Aaron J. Cowieson: Conceptualization, Writing – review & editing.

Disclosures

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.