High Doses of Phytase Alleviate the Negative Effects of Calcium and Phosphorus Imbalance on Growth Performance and Bone Mineralization in Broiler Chickens

ABSTRACT

This study investigated the effect of calcium (Ca) and phytase interaction on growth performance and bone quality in 1-42-day-old broiler chickens. A total of 624 female one-day-old Ross 308 broilers were allotted to 13 treatments with four replicates and 12 birds per replicate. A 2 × 6 factorial experiment was designed to test the combinations of 0.50% and 1.00% Ca with 0, 500, 1,000, 2,500, 5,000, and 10,000 FTU/kg phytase in the basal diet (0.25% non-phytate phosphorus, NPP). The control diet contained adequate Ca and phosphorus (P). Dietary Ca, phytase, and their interaction affected growth performance and bone mineralization of broilers at 1-42 days of age (p<0.05). The broilers fed with 1.00% Ca had lower body weight gain (BWG) and feed intake (FI) compared with the birds fed with 0.50% Ca (p<0.05). The BWG, FI, leg bone weight, and ash weight of the broilers fed with 0.25% NPP were lower than those of birds fed with the control diet (p<0.05). The addition of 500-10,000 FTU/kg phytase improved growth rate and leg bone quality, especially at 1.00% Ca (p<0.05). No differences were observed in growth performance and bone quality of 42-day-old broilers fed with 1.00% Ca + 2,500-10,000 FTU/kg phytase and the control diet (p>0.05). These data indicated that high doses of phytase (2,500-10,000 FTU/kg) alleviate the negative effects of Ca and P imbalance (Ca-to-NPP ratio = 4.0) on growth performance and bone mineralization of broiler chickens.

Keywords:
Calcium; phytase; growth; bone; broiler chicken

INTRODUCTION

Calcium (Ca) and phosphorus (P) are essential minerals in poultry diets. Dietary Ca or P deficiency results in poor growth performance and bone quality in broiler chickens (Han et al., 2018; Li et al., 2020). Limestone, dicalcium phosphate, and monocalcium phosphate are used as feed additives to meet the Ca and P requirements of poultry in China. In poultry feed, total P includes organic P (i.e., phytate P) and inorganic P (i.e., non-phytate P, NPP). NPP is calculted as: NPP = total P − phytate P. NPP is easily used by poultry, whereas phytate P can’t be used effectively.

The optimal dietary Ca-to-NPP ratio is approximately 2.0 in broilers (Rama Rao et al., 2007; Han et al., 2016; Diaz-Alonso et al., 2019), in which dietary Ca and P are considered balanced, and birds obtain greater growth performance. The recommended dietary Ca and NPP levels for 1-21-day-old broilers are 1.00% and 0.45%, respectively (NRC, 1994). In Ca- and P-balanced diet, decreasing the Ca and NPP levels to 0.76% and 0.38% maximizes growth performance of broilers at 1-24 days without negative effects on bone ash and strength (Kiani & Taheri, 2020). These data revealed that Ca and P contents can be appropriately reduced in the balanced diets of broilers.

Dietary Ca and P imbalance has negative effects on growth performance and bone mineralization of broilers (Li et al., 2012). An increase in Ca-to-NPP ratio from 2.1 to 3.8 decreases the BWG and FI of broilers fed with P-deficient diets (Qian et al., 1997). Low P diets are formulated in poultry production to reduce P pollution to the environment. However, the question is how to maintain the growth performance of the broilers fed with P-deficient diets.

Approximately 67% of total P in cereals is presented as phytate P, which can not be effectively utilized by broilers (Steiner et al., 2007). Phytase is used to hydrolyze phytate P in poultry diets. Two kinds of phytase (i. e., endogenous and exogenous phytases) have been reported in poultry. Endogenous phytase is obtained from poultry intestinal mucosa (Maenz & Classen, 1998; Morgan et al., 2015), whereas exogenous phytase is obtained from commercial microbial product, cereal, and its by-products (Xiong et al., 2005; Steiner et al., 2007). Intestinal endogenous phytase can hydrolyze phytate P in broiler diets (Applegate et al., 2003; Tamim et al., 2004). However, only a small amount of phytate P is degraded by endogenous intestinal phytase. Thus, exogenous phytase should be added to poultry diets. In recent years, commercial microbial phytase has been produced and widely used in poultry feed. The addition of phytase increases phytate P hydrolysis and total P retention and improves growth performance and bone quality in broilers fed with P-deficient diets (Shirley & Edwards, 2003; Augspurger & Baker, 2004; Manangi & Coon, 2008). However, the effects of phytase supplementation on growth performance of broilers fed with diets with balanced and imbalanced Ca-to-NPP ratios has not been examined.

Therefore, this study aimed to investigate the effects of dietary phytase levels on growth performance, bone mineralization, and blood mineral concentration in 1- to 42-day-old broilers fed with Ca-inadequate and -adequate diets.

MATERIAL AND METHODS

Animals, diet, and management

All animal experimental procedures used in the present study were approved by the Animal Care and Use Committee of Henan Agricultural University and Shangqiu Normal University.

Phytase was supplied by Guangdong VTR Bio-Tech Co., Ltd. (Zhuhai, China). Microbial phytase was obtained from the Trichoderma strain and expressed in yeast Pichia pastoris. The product contained 5,000 FTU/g phytase, where 1 FTU is equivalent to 1 phytase unit, which represents the amount of enzyme that liberates 1 µmol P per min from 0.0051 mol/L sodium phytate at 37 °C and pH 5.50. The enzyme was added to the diets in powder form.

On the day of hatch, a total of 624 female Ross 308 broilers were randomly allotted to 13 treatment groups with four stainless-steel replicate cages (190 cm × 50 cm × 35 cm) and 12 birds per replicate. A 2 × 6 factorial experiment was designed to test the combinations of 0.50% and 1.00% Ca with 0, 500, 1,000, 2,500, 5,000, and 10,000 FTU/kg phytase in the basal diet containing 0.25% NPP (Table 1). The control diet contained 1.00% Ca and 0.45% NPP for broilers aged 1-21 days and 0.90% Ca and 0.35% NPP for birds aged 22-42 days. The boilers were provided access to mash feed and water ad libitum. The lighting program consisted of 23 h of light and 1 h of darkness on days 1-3, 20 h of light and 4 h of darkness on days 4-21, and 18 h of light and 6 h of darkness on days 22-42. Room temperature was controlled at 33 °C on days 1-3, 30 °C on days 4-7, 27 °C on days 8-21, and 24 °C on days 22-42.

Table 1
Ingredients and nutrient composition of the basal diets.

Item	Days 1-21				Days 22-42
	Control	0.50% Ca	1.00% Ca		Control	0.50% Ca	1.00% Ca
Ingredient (%)
Corn	56.97	60.73	57.91		62.31	65.01	62.19
Soybean meal (43% CP)	32.00	32.00	32.00		28.00	28.00	28.00
Soybean oil	1.60	1.60	1.60		2.60	2.60	2.60
Swine lard	1.32	0.00	0.99		0.95	0.00	0.99
Soybean protein concentrate (63% CP)	3.99	3.47	3.86		2.67	2.30	2.69
Limestone	1.36	0.67	2.09		1.46	0.70	2.13
Dicalcium phosphate	1.94	0.71	0.73		1.35	0.73	0.74
L-lysine·HCl (98%)	0.14	0.14	0.14		0.14	0.14	0.14
DL-methionine (98%)	0.14	0.14	0.14		0.08	0.08	0.08
Trace mineral premix1	0.01	0.01	0.01		0.01	0.01	0.01
Vitamin premix2	0.03	0.03	0.03		0.03	0.03	0.03
Choline chloride (50%)	0.20	0.20	0.20		0.10	0.10	0.10
Sodium chloride	0.30	0.30	0.30		0.30	0.30	0.30
Total	100.00	100.00	100.00		100.00	100.00	100.00
Nutrients composition (%)
Metabolizable energy (kcal/kg)	2,975	2,975	2,975		3,065	3,065	3,065
Crude protein	21.24	21.24	21.24		19.15	19.15	19.15
Calcium	1.00	0.50	1.00		0.90	0.50	1.00
Analyzed calcium	0.98	0.51	0.95		0.86	0.47	0.94
Total phosphorus	0.69	0.49	0.49		0.57	0.48	0.47
Analyzed total phosphorus	0.64	0.46	0.47		0.55	0.46	0.45
Non-phytate phosphorus	0.45	0.25	0.25		0.35	0.25	0.25

• ¹
  The trace mineral premix provided the following nutrients (per kg of diet): 80 mg iron, 40 mg zinc, 8 mg copper, 60 mg manganese, 0.35 mg iodine, and 0.15 mg selenium.
• ²
  The vitamin premix provided the following nutrients (per kg of diet): 8,000 IU vitamin A, 1,000 IU vitamin D₃, 20 IU vitamin E, 0.5 mg menadione, 2.0 mg thiamine, 8.0 mg riboflavin, 35 mg niacin, 3.5 mg pyridoxine, 0.01 mg vitamin B₁₂, 10.0 mg pantothenic acid, 0.55 mg folic acid, and 0.18 mg biotin.

Sample collection

The broilers were weighed on days 1, 21, and 42. All broilers that died spontaneously during the experiment were weighed, and the weight was used to correct the FI. Two chickens per replicate (eight broilers per treatment) were selected randomly for blood and bone collection. Blood samples (5 mL) were collected into tubes with anticoagulant by cardiac puncture on days 21 and 42, and then were centrifuged for 10 min at 3,000 × g at 20 °C to separate plasma. The broilers were euthanized by cervical dislocation after blood sample collection. The femur, tibia, and metatarsus were excised and frozen at -20 °C.

Sample analysis

Blood Ca and P concentrations were determined using a Shimadzu CL-8000 analyzer (Shimadzu Corp., Kyoto, Japan) following the manufacturer’s instructions. Leg bones were cleaned, placed in a container with ethanol for 48 h to remove water and polar lipids, and then extracted in anhydrous ether for 48 h to remove non-polar lipids (Hall et al., 2003). The bones were dried at 105 °C for 24 h before weighing. Bone ash weight and percentage content were determined by burning the leg bones in a muffle furnace for 48 h at 600 °C. The Ca and total P contents in the diets and bones were determined through the method of Han et al. (2009).

Statistical analysis

Replicate means were used as the experimental units. All data in the 13 treatments was analyzed by using one-way ANOVA procedure of SAS software (SAS Institute, 2002). Two-way ANOVA procedure was used to evaluate the main effect of dietary Ca and phytase interaction. Means were compared using Tukey test for significant probability values (p<0.05).

RESULTS AND DISCUSSION

Growth performance

Dietary Ca, phytase, and their interaction affected the BWG and FI of broiler chickens aged 1-21 and aged 1-42 days (p<0.05), but did not affect the feed conversion ratio (FCR) (p>0.05) (Table 2). The increase in dietary Ca from 0.50% to 1.00% negatively affected growth performance of broilers. The broilers fed with 1.00% Ca (Ca-to-NPP ratio = 4) had lower BWG and FI compared with the birds fed with 0.50% Ca (Ca-to-NPP ratio = 2) (p<0.05). These results were in accordance with those reported by previous research (Applegate et al., 2003; Tamim et al., 2004; Rama Rao et al., 2007; Manangi and Coon, 2008; Amerah et al., 2014; Han et al., 2016), in which the highest growth rate of broiler was observed at dietary Ca-to-NPP ratio of 2.0 and the increase of Ca-to-NPP ratio from 2.0 to 7.0 decreased phytate P degradation, BWG, and the FI in the broilers fed with P-deficient diets.

Table 2
Effects of dietary Ca and phytase levels on growth performance of broiler chickens at 1-42 days of age.¹

Ca (%)	Phytase (FTU/kg)	BWG2 (g)			FI2 (g)			FCR2			Mortality (%)
		Days 1-21	Days 22-42	Days 1-42	Days 1-21	Days 22-42	Days 1-42	Days 1-21	Days 22-42	Days 1-42	Days 1-21	Days 22-42	Days 1-42
Control	0	699ab	1649a	2348ab	1103a	3398ab	4501ab	1.58	2.06	1.92	0	0	0
0.50	0	584d	1520ab	2104cd	912c	3056bc	3968d	1.56	2.01	1.89	5.0	2.5	7.5
0.50	500	676ab	1700a	2376a	1059a	3421ab	4480ab	1.57	2.01	1.89	0	0	0
0.50	1,000	667ab	1728a	2395a	1040ab	3386ab	4426abc	1.56	1.96	1.85	0	0	0
0.50	2,500	699ab	1700a	2399a	1075a	3403ab	4478ab	1.54	2.00	1.87	0	0	0
0.50	5,000	643bcd	1699a	2342ab	1063a	3340ab	4403abcd	1.65	1.97	1.88	0	0	0
0.50	10,000	696ab	1717a	2413a	1096a	3372ab	4468ab	1.57	1.96	1.85	0	0	0
1.00	0	486e	1347b	1833e	788d	2665c	3453e	1.62	1.98	1.89	10.0	7.5	17.5
1.00	500	596cd	1485ab	2081d	951bc	3028bc	3979cd	1.60	2.05	1.92	2.5	0	2.5
1.00	1,000	656abc	1469ab	2125bcd	1014ab	3113ab	4127bcd	1.55	2.12	1.94	0	0	0
1.00	2,500	691ab	1629a	2320abc	1058a	3340ab	4398abcd	1.53	2.05	1.90	0	0	0
1.00	5,000	679ab	1631a	2310abcd	1108a	3180ab	4288bcde	1.63	1.95	1.86	0	0	0
1.00	10,000	723a	1724a	2447a	1109a	3504a	4613a	1.53	2.03	1.89	0	0	0
SEM		9	22	27	14	38	48	0.01	0.01	0.01	0.5	0.5	0.8
Main effect
0.50		661a	1677a	2338a	1041a	3329a	4370a	1.58	1.99	1.87	0.8	0.4	1.3
1.00		639b	1548b	2186b	1005b	3138b	4143b	1.58	2.03	1.90	2.1	1.3	3.3
	0	535d	1434b	1969c	850d	2861b	3710c	1.59ab	2.00	1.89	7.5	5.0	12.5
	500	636c	1593ab	2229b	1005c	3224a	4229b	1.59ab	2.03	1.90	1.3	0	1.3
	1,000	662bc	1599ab	2260b	1027bc	3249a	4276ab	1.55b	2.04	1.90	0	0	0
	2,500	695ab	1665a	2360ab	1066abc	3372a	4438ab	1.53b	2.03	1.88	0	0	0
	5,000	661bc	1665a	2326ab	1086ab	3260a	4346ab	1.64a	1.96	1.87	0	0	0
	10,000	710a	1721a	2430a	1103a	3438a	4540a	1.55b	2.00	1.87	0	0	0
Source of variation
Ca		0.005	<0.001	<0.001	0.004	<0.001	<0.001	0.914	0.115	0.139	0.057	0.361	0.043
Phytase		<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	0.007	0.527	0.856	<0.001	0.012	<0.001
Ca ×Phytase		0.002	0.169	0.003	0.001	0.041	0.007	0.452	0.370	0.512	0.138	0.519	0.035

• ¹
  The control diet contained 1.00% Ca and 0.45% NPP for broilers aged 1-21 days and 0.90% Ca and 0.35% NPP for birds aged 22-42 days. The negative diet contained 1.00% Ca and 0.25% NPP for birds aged 1-42 days.
• ²
  BWG = body weight gain, FI = feed intake, and FCR = feed conversion ratio.
• ^a-e
  Means in the same column without a common superscript differ (p<0.05).

Broilers fed with the negative diet (1.00% Ca, 0.25% NPP) had lower BWG and FI than those fed with the control diet (p<0.05). As an essential mineral, P deficiency decreases the growth rate of broilers (Shirley & Edwards, 2003; Manangi & Coon, 2008; Han et al., 2018). Thus, inorganic P or phytase should be added in the diets to meet the P requirement for growth performance of broilers.

The addition of 500 FTU/kg phytase to 0.50% Ca increased the BWG and FI of broilers (p<0.05). The increase in phytase levels from 500 to 10,000 FTU/kg did not affect the BWG, FI, and FCR (p>0.05). By contrast, the addition of 500-10,000 FTU/kg phytase to 1.00% Ca linearly enhanced the BWG and FI of broilers (p<0.05). No differences were observed in the growth rate of 42-day-old broilers fed with 2,500-10,000 FTU/kg phytase (p>0.05). The growth performances of the broilers fed with 0.50% Ca + 500-10,000 FTU/kg phytase and 1.00% Ca + 2,500-10,000 FTU/kg phytase were equivalent to those of the birds fed with control diet.

The addition of phytase improves growth performance of broiler chickens (Shirley & Edwards, 2003; Farhadi et al., 2017; Pieniazek et al., 2017; McCormick et al., 2017; Gautier et al., 2018; Babatunde et al., 2019). Ca and P in broiler diets are considered balanced when the Ca-to-NPP ratio is approximately 2.0 (Rama Rao et al., 2007; Han et al., 2016). Low levels of phytase improve growth performance of broilers when dietary Ca-to-NPP ratio is 2.0 (Driver et al., 2005; Walk et al., 2012). By contrast, high doses of phytase (5,000-10,000 FTU/kg) are needed to maintain the growth of broilers when dietary Ca-to-NPP ratio is 4.6-7.5 (Shirley & Edwards, 2003; Augspurger & Baker, 2004). Similar results were noted in the present study. No differences were observed in the BWG, FI, and FCR of the broilers fed with 0.50% Ca + 500 FTU/kg phytase, 1.00% Ca + 10,000 FTU/kg phytase, and control diet. Hence, a small amount of phytase (500 FTU/kg) in Ca- and P-balanced diet (Ca-to-NPP ratio = 2.0) is adequate for broiler growth, but high doses of phytase (2,500-10,000 FTU/kg) are needed to alleviate the negative effects of Ca and P imbalance (Ca-to-NPP ratio = 4.0) on growth performance of broilers.

Bone mineralization

Dietary Ca, phytase, and their interaction affected leg bone mineralization of broiler chickens (p<0.05) (Tables 3, 4, and 5). Increasing dietary Ca from 0.50% to 1.00% decreased bone quality in broilers aged 21 days. The percentages of ash and Ca in the femur, tibia, and metetarsus of 21-day-old broilers fed with 1.00% Ca (Ca-to-NPP ratio = 4.0) were lower than those of birds fed with 0.50% Ca (Ca-to-NPP ratio = 2.0) (p<0.05). Our results agreed with those reported by Qian et al. (1997), in which the increase in Ca-to-NPP ratio from 2.1 to 3.8 decreased the toe ash percentage of broilers. Hence, dietary Ca and P imbalance deteriorates the bone development of poultry.

Table 3
Effects of dietary Ca and phytase levels on femur mineralization of broiler chickens at 21 and 42 days of age.

Ca (%)	Phytase (FTU/kg)	Weight (g)		Ash (g)		Ash (%)		Ca (%)		p (%)
		Day 21	Day 42	Day 21	Day 42	Day 21	Day 42	Day 21	Day 42	Day 21	Day 42
Control	0	1.36a	4.30ab	0.64a	1.96a	47.53a	45.87ab	17.03ab	17.08ab	8.72a	9.10a
0.50	0	1.08bcd	3.58bc	0.47cd	1.51bc	45.68ab	42.51d	16.95ab	15.61c	8.31a	7.64b
0.50	500	1.23abc	4.31ab	0.58abc	1.86ab	47.42a	43.61bcd	17.48a	16.00bc	8.56a	8.16ab
0.50	1,000	1.15abcd	4.17ab	0.54abc	1.89ab	46.66a	44.79abcd	17.61a	16.83abc	8.34a	8.29ab
0.50	2,500	1.19abcd	3.87ab	0.54abc	1.76ab	45.83ab	44.91abcd	17.69a	16.54abc	8.36a	8.57ab
0.50	5,000	1.07cd	3.99ab	0.50bc	1.77ab	46.76a	43.60bcd	16.84ab	16.84abc	8.32a	8.33ab
0.50	10,000	1.33ab	3.88ab	0.63ab	1.55bc	47.41a	42.47d	17.13ab	16.26abc	8.53a	8.13ab
1.00	0	0.92d	3.04c	0.34d	1.29c	42.28b	42.69cd	14.80c	15.82bc	6.64c	7.52b
1.00	500	1.04cd	3.58bc	0.45cd	1.57bc	42.81b	44.36abcd	15.58bc	15.97bc	7.31bc	8.03ab
1.00	1,000	1.24abc	3.77abc	0.56abc	1.67ab	45.28ab	44.58abcd	16.24abc	16.34abc	8.08ab	8.01ab
1.00	2,500	1.33ab	3.82abc	0.63ab	1.71ab	47.19a	45.38abc	17.22a	16.58abc	8.42a	8.47ab
1.00	5,000	1.21abc	4.22ab	0.58abc	1.97a	47.06a	46.79a	17.14ab	17.39a	8.47a	8.90a
1.00	10,000	1.38a	4.54a	0.66a	2.05a	48.24a	45.40abc	17.56a	17.10ab	8.44a	9.10a
SEM		0.02	0.07	0.01	0.03	0.31	0.23	0.14	0.10	0.09	0.10
Main effect
0.50		1.17	3.97	0.54	1.73	46.63a	43.65b	17.28a	16.35	8.40a	8.18
1.00		1.19	3.83	0.54	1.71	45.48b	44.87a	16.42b	16.53	7.89b	8.34
	0	1.00c	3.31b	0.40c	1.40b	43.98c	42.60b	15.88b	15.71c	7.48b	7.58b
	500	1.14bc	3.95a	0.51b	1.72a	45.11bc	43.99ab	16.53ab	15.98bc	7.93ab	8.10ab
	1,000	1.19ab	3.97a	0.55b	1.78a	45.97abc	44.68a	16.93a	16.59ab	8.21a	8.15ab
	2,500	1.26ab	3.85a	0.59ab	1.73a	46.51ab	45.14a	17.46a	16.56ab	8.39a	8.52a
	5,000	1.14bc	4.11a	0.54b	1.87a	46.91ab	45.19a	16.99a	17.11a	8.39a	8.61a
	10,000	1.36a	4.21a	0.65a	1.80a	47.82a	43.94ab	17.34a	16.68ab	8.48a	8.61a
Source of variation
Ca		0.663	0.130	0.716	0.715	0.017	<0.001	<0.001	0.231	<0.001	0.935
Phytase		<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	0.002
Ca ×Phytase		0.006	<0.001	<0.001	<0.001	0.002	0.006	<0.001	0.211	<0.001	0.173

• ^a-d
  Means in the same column without a common superscript differ (p<0.05).

Table 4
Effects of dietary Ca and phytase levels on tibia mineralization of broiler chickens at 21 and 42 days of age.

Ca (%)	Phytase (FTU/kg)	Weight (g)		Ash (g)		Ash (%)		Ca (%)		p (%)
		Day 21	Day 42	Day 21	Day 42	Day 21	Day 42	Day 21	Day 42	Day 21	Day 42
Control	0	1.79ab	5.89ab	0.86ab	2.66ab	48.00ab	47.37ab	17.55ab	17.57ab	8.82ab	8.53abcd
0.50	0	1.32ef	5.07bcd	0.61d	2.29bc	46.26b	45.17bc	16.67abc	16.02c	7.87bc	7.71d
0.50	500	1.54bcde	5.95ab	0.72bcd	2.65ab	46.97ab	45.42bc	16.85abc	16.40bc	8.53ab	7.92cd
0.50	1,000	1.60abcd	5.50bc	0.75abcd	2.59ab	47.24ab	48.50a	17.42ab	17.42abc	8.54ab	8.05bcd
0.50	2,500	1.70abc	5.25bc	0.82abc	2.45abc	48.18ab	46.93abc	17.51ab	17.11abc	8.00ab	8.17abcd
0.50	5,000	1.40de	5.11bcd	0.68cd	2.41abc	47.68ab	48.41a	17.44ab	16.48abc	8.43ab	8.02bcd
0.50	10,000	1.66abc	5.30bc	0.79abc	2.46abc	47.69ab	46.72abc	17.43ab	16.80abc	8.12ab	8.95ab
1.00	0	1.13f	4.16d	0.44e	1.86c	38.65d	44.79c	14.56d	16.55abc	6.68c	8.10bcd
1.00	500	1.47cde	4.75cd	0.63d	2.23bc	43.11c	47.24ab	16.04c	17.59ab	7.63bc	8.62abcd
1.00	1,000	1.62abcd	4.99bcd	0.75abcd	2.31bc	47.22ab	46.27abc	17.94a	17.60ab	8.75ab	8.54abcd
1.00	2,500	1.81a	5.29bc	0.83abc	2.48abc	47.55ab	47.07abc	16.47bc	17.51abc	7.85bc	9.02ab
1.00	5,000	1.60abcd	5.60bc	0.79abc	2.58ab	47.77ab	46.19abc	17.01abc	17.41abc	8.80ab	9.12a
1.00	10,000	1.84a	6.76a	0.87a	3.01a	48.68a	48.15a	17.98a	17.93a	9.11a	8.88abc
SEM	3.4	0.03	0.10	0.02	0.05	0.38	0.20	0.14	0.11	0.11	0.10
Main effect
0.50		1.53	5.36	0.73	2.48	47.34a	46.86	17.22a	16.70b	8.25	8.14b
1.00		1.58	5.26	0.72	2.41	45.50b	46.62	16.67b	17.43a	8.14	8.72a
	0	1.22c	4.62c	0.53d	2.07b	42.45c	44.98b	15.62c	16.29b	7.27b	7.91b
	500	1.50b	5.35b	0.68c	2.44ab	45.04b	46.33ab	16.44b	16.99ab	8.08a	8.27ab
	1,000	1.61ab	5.25b	0.75bc	2.45ab	47.23a	47.38a	17.68a	17.51a	8.65a	8.30ab
	2,500	1.76a	5.27b	0.82ab	2.46a	47.86a	47.00a	16.99ab	17.31a	7.93ab	8.59ab
	5,000	1.50b	5.35b	0.74c	2.50a	47.72a	47.30a	17.23a	16.94ab	8.62a	8.57ab
	10,000	1.75a	6.03a	0.83a	2.74a	48.19a	47.43a	17.70a	17.36a	8.62a	8.92a
Source of variation
Ca		0.178	0.389	0.520	0.402	<0.001	0.381	<0.001	<0.001	0.431	0.002
Phytase		<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	0.004	<0.001	0.034
Ca ×Phytase		0.004	<0.001	<0.001	0.003	<0.001	<0.001	<0.001	0.478	0.001	0.466

• ^a-f
  Means in the same column without a common superscript differ (p<0.05).

Table 5
Effects of dietary Ca and phytase levels on metatarsus mineralization of broiler chickens at 21 and 42 days of age.

Ca (%)	Phytase (FTU/kg)	Weight (g)		Ash (g)		Ash (%)		Ca (%)		p (%)
		Day 21	Day 42	Day 21	Day 42	Day 21	Day 42	Day 21	Day 42	Day 21	Day 42
Control	0	1.02a	3.27a	0.47a	1.36a	46.12a	41.61ab	16.83ab	15.14ab	8.11a	7.40
0.50	0	0.80abc	2.80ab	0.35bc	1.16ab	43.58abc	41.79ab	16.64ab	15.18ab	7.54ab	7.45
0.50	500	0.91ab	3.27a	0.41abc	1.40a	44.96ab	42.79ab	16.88ab	15.57ab	8.02a	7.82
0.50	1,000	0.91ab	2.90ab	0.41abc	1.24ab	45.58ab	42.81ab	16.94ab	16.29ab	8.10a	7.76
0.50	2,500	0.91ab	2.92ab	0.42abc	1.28ab	45.64ab	44.17a	16.80ab	16.39a	8.07a	7.89
0.50	5,000	0.78bc	2.96ab	0.36bc	1.28ab	45.80a	43.42ab	16.66ab	16.28ab	8.16a	8.06
0.50	10,000	0.95ab	2.94ab	0.44abc	1.19ab	46.03a	40.67b	17.04ab	15.38ab	8.15a	7.62
1.00	0	0.66c	2.39b	0.24d	1.00b	36.33d	41.81ab	12.90d	15.23ab	5.85c	7.43
1.00	500	0.84abc	2.78ab	0.34c	1.13ab	40.54c	41.50ab	14.53cd	14.69b	6.77b	7.64
1.00	1,000	0.99ab	3.08a	0.41abc	1.25ab	41.83bc	40.79ab	15.44bc	15.45ab	7.08b	7.75
1.00	2,500	0.97ab	3.03ab	0.43abc	1.24ab	44.50ab	40.99ab	16.57ab	15.46ab	7.56ab	7.60
1.00	5,000	0.94ab	3.22a	0.44ab	1.34a	44.79ab	41.68ab	15.96abc	15.91ab	7.35ab	8.05
1.00	10,000	1.02a	3.34a	0.48a	1.37a	47.38a	41.71ab	17.36a	15.45ab	8.07a	7.52
SEM		0.02	0.05	0.01	0.02	0.44	0.22	0.19	0.11	0.10	0.05
Main effect
0.50		0.88	2.96	0.40	1.26	45.26a	42.61a	16.83a	15.85a	8.01a	7.77
1.00		0.90	2.97	0.39	1.22	42.56b	41.41b	15.46b	15.37b	7.11b	7.67
	0	0.73b	2.59b	0.29c	1.08b	39.96d	41.80	14.77c	15.20	6.70c	7.44b
	500	0.88a	3.03a	0.37b	1.27a	42.75c	42.14	15.71bc	15.13	7.40b	7.73ab
	1,000	0.95a	2.99ab	0.41ab	1.25ab	43.70bc	41.80	16.19ab	15.87	7.59ab	7.76ab
	2,500	0.94a	2.97ab	0.42ab	1.26a	45.07abc	42.58	16.69ab	15.93	7.82ab	7.75ab
	5,000	0.86ab	3.09a	0.40b	1.31a	45.29ab	42.55	16.31ab	16.09	7.75ab	8.06a
	10,000	0.98a	3.14a	0.46a	1.28a	46.70a	41.19	17.20a	15.42	8.11a	7.57ab
Source of variation
Ca		0.270	0.899	0.623	0.273	<0.001	0.003	<0.001	0.022	<0.001	0.314
Phytase		<0.001	0.005	<0.001	0.007	<0.001	0.269	<0.001	0.034	<0.001	0.024
Ca ×Phytase		0.016	0.008	<0.001	0.008	<0.001	0.033	<0.001	0.495	0.002	0.950

• ^a-d
  Means in the same column without a common superscript differ (p<0.05).

Dietary P-deficiency influenced bone development. The bone weight, ash weight, and percentage content of ash in the femur and tibia of 42-day-old broilers fed the negative diet (1.00% Ca and 0.25% NPP) were lower than those of birds fed with the control diet (p<0.05). These results were in accordance with those reported by Viveros et al. (2002), Han et al. (2018), and Li et al. (2020), in which P-deficient diet caused lower tibia weight, ash weight, and percentage contents of ash, Ca, and P in broilers. Broilers are sensitive to insufficient P in diets. P deficiency damages the bone growth and mineralization of broilers.

The addition of phytase did not enhance bone weight and ash weight in the femur, tibia, and metatarsus of 42-day-old broilers at 0.50% Ca (p>0.05). By contrast, adding phytase improved leg bone development at 1.00% Ca (p<0.05). The bone weight and ash weight in the three leg bones of broilers at 42 days were linearly increased by dietary phytase levels at 1.00% Ca (p<0.05). The interactions between dietary Ca and phytase were observed in the weight, ash weight, and ash percentage content of the leg bones in 42-day-old broilers (p<0.05). No differences in bone weight and ash weight were observed among the broilers fed with 0.50% Ca + 500-5,000 FTU/kg phytase, 1.00% Ca + 2,500-10,000 FTU/kg phytase, and the control diet (p>0.05).

The addition of phytase improves tibia ash weight and percentage in broilers fed with low-P diets (Shirley & Edwards, 2003; Augspurger & Baker, 2004; Han et al., 2009; Walk et al., 2012; Pieniazek et al., 2017; McCormick et al., 2017; Gautier et al., 2018). In the present study, the addition of phytase did not affect the leg bone mineralization of 42-day-old broilers at 0.50% Ca. By contrast, phytase linearly improved the bone quality of the tibia, femur, and metatarsus of broilers at 1.00% Ca. These data revealed that the efficacy of phytase is affected by the Ca-to-NPP ratio, and high doses of phytase (2,500-10,000 FTU/kg) are required to alleviate the negative effect of dietary Ca and P imbalance on the bone mineralization of broilers.

Blood mineral concentration

Dietary Ca, phytase, and their interaction affected blood P concentration in broilers at 21 days of age (p<0.05) but did not affect blood Ca concentration (p>0.05) (Table 6). The broilers fed with 1.00% Ca had higher plasma Ca concentration but lower plasma P concentration than the birds fed with 0.50% Ca (p<0.05). Dietary Ca level affects the blood mineral concentration of broilers (Han et al., 2016; Li et al., 2020), in which increasing the Ca level enhanced blood Ca but decreased blood P concentration in 21-day-old chickens. These data indicated that high dietary Ca-to-NPP ratio resulted in blood Ca and P imbalance.

The plasma P concentration in 21-day-old broilers fed with the negative diet (1.00% Ca and 0.25% NPP) was lower than that in the birds fed with the control diet (p<0.05). These results were in accordance with those reported by previous research (Han et al., 2009; Li et al., 2020), in which P deficiency decreased the blood P concentration, and supplemental P restored the blood P concentration of broilers.

The addition of phytase increased blood P concentration in 21-day-old broilers at 1.00% Ca (p<0.05). Phytase did not affect plasma Ca and P concentrations in 42-day-old broilers (p>0.05). Our results agreed with those reported by Viveros et al. (2002), Shirley & Edwards (2003), and Han et al. (2009), in which the addition of phytase increased plasma P concentration of broilers fed with P-deficient diet. Broilers grow fast from hatching to growth phase. A large amount of P is needed to maintain growth rate. Blood mineral concentration is sensitive to phytase addition. By contrast, the mineral metabolism of broilers is relatively stable from grower to finisher phase and blood mineral concentration can be balanced through self-regulation.

Table 6
Effects of dietary Ca and phytase levels on plasma mineral concentration in broiler chickens.

Ca (%)	Phytase (FTU/kg)	Ca (mg/100mL)			p (mg/100mL)
		Day 21	Day 42		Day 21	Day 42
Control	0	9.05ab	10.31		6.72ab	7.48
0.50	0	8.00b	10.20		6.56ab	7.85
0.50	500	9.04ab	9.65		6.66ab	7.82
0.50	1,000	9.04ab	10.33		6.91a	8.02
0.50	2,500	9.16ab	9.71		6.52ab	7.05
0.50	5,000	10.02ab	9.20		6.20ab	7.15
0.50	10,000	9.58ab	10.37		5.91ab	7.46
1.00	0	11.38a	9.47		3.68c	7.73
1.00	500	9.87ab	9.31		5.26b	7.37
1.00	1,000	8.69ab	10.32		6.74ab	7.81
1.00	2,500	8.59ab	10.43		6.81a	7.66
1.00	5,000	8.98ab	9.68		6.98a	7.27
1.00	10,000	8.93ab	9.81		6.59ab	6.97
SEM		0.18	0.12		0.14	0.13
Main effect
0.50		9.14	9.91		6.46a	7.56
1.00		9.41	9.84		6.01b	7.47
	0	9.69	9.84		5.12b	7.79
	500	9.46	9.48		5.96ab	7.59
	1,000	8.87	10.33		6.82a	7.91
	2,500	8.88	10.07		6.67a	7.35
	5,000	9.50	9.44		6.59a	7.21
	10,000	9.26	10.09		6.25a	7.21
Source of variation
Ca		0.446	0.784		0.016	0.767
Phytase		0.667	0.339		<0.001	0.623
Ca ×Phytase		0.008	0.570		<0.001	0.889

• ^a-c
  Means in the same column without a common superscript differ (p<0.05).

CONCLUSIONS

In conclusion, low doses of phytase (500-1,000 FTU/kg) are adequate for growth and bone development of the broilers fed with Ca- and P-balanced diets (0.50% Ca, 0.25% NPP, and Ca-to-NPP ratio = 2.0). However, high doses of phytase (2,500-10,000 FTU/kg) are needed to alleviate the negative effects of Ca and P imbalance (1.00% Ca, 0.25% NPP, and Ca-to-NPP ratio = 4.0) on growth performance and bone mineralization of broiler chickens from 1 to 42 days of age.

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• FUNDING

  This work was supported by the National Natural Science Foundation of China (32072753 and U1704107) and the Key Scientific Research Rrojects of Universities in Henan Province (16A230014).

Publication Dates

• Publication in this collection
  21 Nov 2022
• Date of issue
  2022

History

• Received
  13 Sept 2021
• Accepted
  04 May 2022