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

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.


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 byproducts (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 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 1to 42-day-old broilers fed with Ca-inadequate and -adequate diets.

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.

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

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

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.

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

High Doses of Phytase Alleviate the Negative Effects of Calcium and Phosphorus Imbalance on Growth Performance and Bone Mineralization in Broiler Chickens
is 4.6-7.5 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.
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).

High Doses of Phytase Alleviate the Negative Effects of Calcium and Phosphorus Imbalance on Growth Performance and Bone Mineralization in Broiler Chickens
The addition of phytase improves tibia ash weight and percentage in broilers fed with low-P diets 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), , 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

High Doses of Phytase Alleviate the Negative Effects of Calcium and Phosphorus Imbalance on Growth Performance and Bone Mineralization in Broiler Chickens
phase and blood mineral concentration can be balanced through self-regulation.