key: cord-0798402-00c1ehhu
authors: Cao, Sumei; Li, Tingting; Shao, Yuxin; Zhang, Liyang; Lu, Lin; Zhang, Rijun; Hou, Shuisheng; Luo, Xugang; Liao, Xiudong
title: Regulation of bone phosphorus retention and bone development possibly by related hormones and local bone-derived regulators in broiler chicks
date: 2021-08-12
journal: J Anim Sci Biotechnol
DOI: 10.1186/s40104-021-00610-1
sha: 3bdf846ca1de7023e8ef89fc974b9c99780e64ef
doc_id: 798402
cord_uid: 00c1ehhu

BACKGROUND: Phosphorus is essential for bone mineralization in broilers, however, the underlying mechanisms remain unclear. We aimed to investigate whether bone phosphorus retention and bone development might be regulated by related hormones and local bone-derived regulators in broilers. METHODS: Broilers were fed diets containing different levels of non-phytate phosphorus (NPP) 0.15%, 0.25%, 0.35%, 0.45% and 0.55% or 0.15%, 0.22%, 0.29%, 0.36% and 0.43% from 1 to 21 or 22 to 42 days of age. Serum and tibia samples were collected for determinations of bone phosphorus retention and bone development parameters, related hormones and local bone-derived regulators of broiler chickens on d 14, 28 and 42, respectively. RESULTS: Tibia ash phosphorus, total phosphorus accumulation in tibia ash (TP(TA)), bone mineral concentration (BMC), bone mineral density (BMD), bone breaking strength (BBS), and ash on d 14, 28 or 42, serum 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) on d 28 and 42, mRNA expressions of tibia fibroblast growth factor 23 (FGF23) and dentin matrix protein 1 (DMP1) on d 14 and 28 increased linearly or quadratically (P < 0.05), while serum parathyroid hormone (PTH) on d 28, tibia alkaline phosphatase (ALP) on d 14, 28 and 42, bone gal protein (BGP) on d 14, and mRNA expression of tibia phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) on d 14 and 28 decreased linearly or quadratically (P < 0.04) as dietary NPP level increased. TP(TA,) BMC, BMD, and ash on d 28 and 42, BBS on d 28, and ash phosphorus on d 42 were positively correlated (r = 0.389 to 0.486, P < 0.03) with serum 1,25(OH)(2)D(3). All of the above parameters were positively correlated (r = 0.380 to 0.689, P < 0.05) with tibia DMP1 mRNA expression on d 14, 28 and 42, but negatively correlated (r = − 0.609 to − 0.538, P < 0.02) with serum PTH on d 28, tibia ALP on d 14, 28 and 42, and BGP on d 14. TP(TA), BMC and ash on d 14 and BMD on d 28 were negatively correlated (r = − 0.397 to − 0.362, P < 0.03) with tibia PHEX mRNA expression, and BMD on d 28 was positively correlated (r = 0.384, P = 0.04) with tibia FGF23 mRNA expression. CONCLUSIONS: These results suggested that bone phosphorus retention and bone development parameters had moderate to strong correlations with serum PTH and 1,25(OH)(2)D(3) and tibia DMP1, PHEX, FGF23, ALP and BGP in broilers during the whole growth period, and thus they might be partly regulated by these related hormones and local bone-derived regulators. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40104-021-00610-1.

Conclusions: These results suggested that bone phosphorus retention and bone development parameters had moderate to strong correlations with serum PTH and 1,25(OH) 2 D 3 and tibia DMP1, PHEX, FGF23, ALP and BGP in broilers during the whole growth period, and thus they might be partly regulated by these related hormones and local bone-derived regulators.

Keywords: Bone, Broiler chicken, Hormone, Local bone-derived regulator, Phosphorus Background Phosphorus, in the form of inorganic phosphorus, is an essential mineral element and plays an important role in multiple biological processes, especially for bone mineralization in humans and animals [1, 2] . Many clinical researches have demonstrated that phosphorus is one of the major factors in the maintenance of bone development and health, and the deficiency of phosphorus could result in bone pathology and clinical illness [3, 4] . Besides, broiler chickens are fast-growing birds and susceptible to dietary phosphorus deficiency, and early fast growth rate is generally accompanied by a high incidence of skeletal disorders, such as hypophosphatemic rickets and tibial dyschondroplasia, partly due to an insufficient formation of hydroxyapatite in the bone of broiler chickens [5] . Thus, broiler chickens might be an ideal animal model for studying the bone development and health, and it is necessary to study bone phosphorus retention and bone development for better improving phosphorus utilization and homeostasis in the bone of broiler chicken model. The phosphorus homeostasis is achieved by complex interactions between bone and multiple organs via many hormones including but not limited to parathyroid hormone (PTH), 1, 25dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) and fibroblast growth factor 23 (FGF23), and most of these insights are derived from human genetic disorders and genetically engineered mice [6, 7] . Furthermore, many recent studies have shown that local bone-derived factors, such as FGF23, phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX), matrix extracellular phosphoglycoprotein (MEPE) and dentin matrix protein 1 (DMP1), constitute a novel hormone/ enzyme/extracellular matrix protein axis that regulates phosphorus homeostasis and bone mineralization [8] [9] [10] . Additionally, bone is a living tissue that is constantly changing and adapting during growth by osteoblasts and osteocytes, and osteoblasts is usually considered as a major factor regulating the skeleton growth, remodeling, and mineralization [11, 12] . It is crucial to maintain adequate inorganic phosphorus levels in the process of matrix mineralization by osteoblasts and osteocytes, and alkaline phosphatase (ALP) and bone gal protein (BGP) are also involved in the process of bone formation and matrix mineralization [13, 14] . It is thus a complex interaction process between the related hormones and local bone-derived factors for the regulation of bone phosphorus retention and bone development in the bone and needs to be further studied with animal models. However, only a few studies have been conducted to evaluate the regulation of bone phosphorus retention and bone development by FGF23 in chickens [15] [16] [17] . A recent study from our laboratory showed that the bone phosphorus retention was involved in the bone development of broilers fed the diets containing the fixed levels of non-phytate phosphorus (NPP) at different ages during d 1 to 42 possibly via the regulation of the bone ALP and BGP [12] . To our knowledge, no study has been undertaken to investigate how PHEX, MEPE, DMP1, ALP and BGP regulate bone phosphorus retention and bone development of broiler chickens fed diets containing different NPP levels from 1 to 42 days of age. Therefore, possible mechanisms of bone phosphorus retention and bone development of broilers during the whole growth period remain unclear. It was hypothesized that bone phosphorus retention and bone development of broiler chickens during the whole growth period might be regulated not only by the related hormones, but also by the local bone-derived factors in broiler chickens. The objective of the present study was to investigate the effect of dietary different levels of NPP on the growth performance, bone phosphorus retention and bone development parameters, related hormones and local bone-derived regulators in the fast-growing broiler chicken model to test the above hypothesis.

All experimental procedures were approved by the Animal Management Committee (in charge of animal welfare) of the Institute of Animal Science, Chinese Academy of Agricultural Sciences (IAS-CAAS, Beijing, China) and performed in accordance with the guidelines. Ethical approval on animal survival was given by the animal ethics committee of IAS-CAAS. We have followed the ARRIVE guidelines for reporting animal research [18] .

A total of 800 one-d-old Arbor Acres male broiler chicks (Huadu Broiler Breeding Corp., Luanping, China) with similar body weight were randomly allotted to 1 of 5 treatments with 8 replicate cages of 20 chicks per replicate cage. The birds were housed in electrically heated, thermostatically controlled cages (190 cm × 100 cm × 75 cm, length × width × height) equipped with the plastic net floor and fiberglass feeders (180 cm, length) and water supply in an environmentally controlled room with continuous 18 h of light and 6 h of dark. Environmental temperature was maintained at 35°C for the first 3 d, after which the temperature was gradually reduced by 3°C per week until it reached 24°C and then was maintained at this temperature until the end of the experiment. The light intensity of 20 lx was used during the entire experimental period. Feed and tap water were provided ad libitum. Mortality was recorded daily, and chick weight and feed intake per cage were measured at 21 and 42 days of age to calculate average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR).

A completely randomized design was used in this experiment. There were a total of 5 treatments of dietary NPP levels, including 0.15%, 0.25%, 0.35%, 0.45% (control group) and 0.55% or 0.15%, 0.22%, 0.29%, 0.36% (control group) and 0.43% for broilers from 1 to 21 days of age or 22 to 42 days of age according to the National Research Council (NRC, 1994) requirements of broilers for dietary NPP levels (d 1-21: 0.45%, and d 22-42: 0.35%) [19] , respectively.

The basal corn-soybean meal diets were formulated to meet or exceed the nutrient requirements of starter broilers (d 1 to 21) and grower broilers (d 22 to 42) as recommended by the Feeding standard of chicken (China, NY/T 2004) [20] [21, 22] according to the above experimental design and then mixed with each aliquot of the basal diet. Fine sand was washed several times before use, and contained no detectable phosphorus and Ca. All diets were fed in mash form. Dietary composition and nutrient levels are all expressed on an as-fed basis and are presented in Supplementary Table S1 .

The feed ingredients and diets from the 5 treatments were collected for analyses of crude protein (CP), Ca and phosphorus before the initiation of the experiment to confirm CP, Ca, and phosphorus concentrations in diets. At 14, 28, and 42 days of age, 4, 3 or 2 birds respectively from each replicate cage were selected according to average body weight of each cage after a 12-h fast. Blood samples were taken from the selected birds via the wing vein, immediately placed on ice, and then centrifuged at 3,000×g for 10 min at 4°C to isolate serum, and then serum aliquots were stored at − 20°C until analyses. The selected birds were then killed by cervical dislocation, the right tibia was peeled and frozen at − 20°C and the left tibia was peeled and frozen in liquid nitrogen and then stored at − 80°C until analyses. To reduce individual biological variation, the samples from selected birds in each replicate cage were pooled into 1 sample in equal ratios before analyses. Therefore, the number of replication was normally 8 (n = 8) for each parameter measurement of each treatment.

The Ca concentrations in feed ingredient, diet and tibia ash samples were measured using an inductively coupled plasma emission spectroscope (model IRIS Intrepid II, Thermal Jarrell Ash, Waltham, MA) after wet digestions with HNO 3 and HClO 4 [23, 24] . The phosphorus concentrations in feed ingredients, diet samples and tibia ash were measured colorimetrically using the vanadatemolybdate method [25] . The phytate phosphorus (PP) concentrations of feed ingredients and diets were analyzed with the ferric precipitation method as described by Rutherfurd et al. [26] and then the dietary NPP was calculated with the formula to total phosphorus -PP. The concentrations of CP in feed ingredient and diet samples were determined using the methods of Association of Official Analytical Chemists (1990) [27] .

Determination of the tibia bone mineral concentration, bone mineral density, bone breaking strength, ash concentrations and the total phosphorus accumulation in tibia ash

The frozen tibia was thawed at room temperature for 2 h and then stripped of all soft tissues. The tibia bone mineral concentration (BMC) and bone mineral density (BMD) were then determined using Dual-Energy X-Ray Absorptiometry (DEXA, Tokyo, Japan) [12, 28] . The tibia bone breaking strength (BBS) of broilers on d 14, 28, and 42 was determined by a 3-point bending test (HDP/3 PB Texture Analyzers, UK) [12, 29] . To determine tibia ash concentration, the tibia was dried at 105°C for 24 h, and then defatted with fresh diethyl ether for 48 h, and finally the fat-free, dried bone was ashed in a muffle furnace at 550°C for 16 h [28] . The total phosphorus accumulation in tibia ash (TP TA ) was calculated as described in our previous study [12] .

Determination of serum ca, phosphorus, ALP, FGF23, PTH, and 1,25(OH) 2 D 3 concentrations and tibia ALP activity and BGP concentration Serum was thawed and analyzed for the Ca concentration using a microplate reader with Ca assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China); serum phosphorus concentration was determined using the molybdenum blue method according to Goldenberg and Fernandez [30] ; serum intact FGF23 concentration was assessed using an ELISA kit specially used for poultry according to the manufacturer's protocol (Nanjing Jiancheng Bioengineering Institute, Nanjing, China); serum PTH level was measured using an ELISA kit according to the manufacturer's protocol (Nanjing Jiancheng Bioengineering Institute, Nanjing, China); and serum 1,25(OH) 2 D 3 level was measured by an ELISA kit by Beijing Sino-UK Institute of Biological Technology. The ALP activities in serum and tibia were measured using a microplate reader with ALP assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The tibia BGP concentration was determined by the method of ELISA with a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Total RNA from the tibia bone was isolated using TRIzol reagent (catalogue no. 15596018; Life Technologies) and reversely transcribed into complementary DNA (cDNA) according to the manufacturer's instructions. The concentration of total RNA was estimated by measuring its optical density at 260 and 280 nm with a spectrophotometer (ND-100; NanoDrop Technologies), and the purity was determined in agarose gels stained with ethidium bromide. Then 500 ng of total RNA was reversely transcribed into cDNA using PrimeScript TM RT Master Mix (catalogue no. RR047A; TaKaRa Bio Inc.) according to the manufacturer's instructions. cDNA was used as templates for real-time quantitative PCR machine following the manufacturer's instructions. The PCR protocol was as follow: denaturation at 95°C for 2 min followed by 40 cycles of 40 cycles of 94°C for 15 s and 60°C for 1 min. The primer sequences are listed in Supplementary Table S2 . The mRNA level of the target gene was calculated using the 2 -△△Ct method, and the geometric mean of internal references, β-actin and glyceraldehydes 3-phosphate dehydrogenase (GAPDH) were used to normalize the expression of the targeted genes [31, 32] .

Tibia protein was isolated from liquid nitrogen snap frozen tibia by pulverizing the bone using a mortar and pestle and transferring the powder (40 mg) to 0.4 mL of ice-cold radio immunoprecipitation assay (RIPA) lysis buffer (catalogue no. P0013B, Beyotime Institute of Biotechnology) supplemented with 4 μL of protease inhibitor (catalogue no. B14001; BioTool). The lysates were sonicated for 10 s and 8 times in an ice-cold water bath and centrifuged to separate the protein extract, and the supernatant was collected for total protein determination using a bicinchoninic acid protein assay kit (catalogue no. 23225; Pierce). The extracted protein (30 μg) was subjected to electrophoresis on a 10% SDS-PAGE gel, and then electrotransferred onto the polyvinylidenefluoride membranes (catalogue no. IVPH00010; Merck-Millipore). After the transfer, membranes were blocked for 1 h at room temperature in a blocking buffer with 5% skimmed milk, and then incubated overnight at 4°C with the following primary antibodies: FGF23 (catalogue no. A6124; Abclonal), PHEX (catalogue no. A7918; Abclonal), MEPE (catalogue no. ab108073; abcam), DMP1 (catalogue no. ab103203; abcam) and β-actin (catalogue no. HX1827; Huaxingbio). After several washes in tris-buffered saline with tween, membranes were incubated with the secondary antibody of goat anti-rabbit (catalogue no. HX2030; Huaxingbio) or goat anti-mouse (catalogue no. HX2032; Huaxingbio) for 1 h at room temperature. The signals were recorded with a chemiluminescence image scanner (Tanon, Shanghai, China) by enhanced chemiluminescence using a Super Signal West Pico Trail Kit (catalogue no. 34077; Pierce). Data were presented as the ratio of FGF23, PHEX, MEPE or DMP1 protein band intensity to β-actin protein band intensity. The β-actin protein was used to normalize the expression level of FGF23, PHEX, MEPE or DMP1.

Data from the present study were analyzed by SAS statistical software (version 9.4, SAS Institute Inc., Cary, NC). The data were firstly tested for normality and homogeneity of variance using the Shapiro-Wilk and Ftests, respectively. Then the data were subjected to oneway ANOVA using the general liner model procedure, and the treatment comparisons for significant differences in all figures were tested by the least significant difference method. Orthogonal polynomials were applied for testing linear and quadratic effects of dependent variables to independent variables. Simple correlation analyses between the bone phosphorus retention or bone development parameters and related hormones or local bone-derived regulators were carried out based on replicate values from different NPP level treatments at each sampling time point using the Pearson procedure [12, 33] . The degree of correlation (strong correlation: ± 0.50 and ± 1; moderate correlation: ± 0.30 and ± 0.49; weak correlation: below ±0.29; and no correlation: 0) was determined as described by Chatterjee et al. [34] . The replicate cage was regarded as an experimental unit. The statistical significance was set at P ≤ 0.05.

Dietary NPP level affected (P < 0.0001) ADG, ADFI, and mortality of broilers from 1 to 21 and 22 to 42 days of age, as well as FCR of broilers from 1 to 21 days of age, but did not affect (P = 0.67) FCR of broilers from 22 to 42 days of age (Fig. 1) . From 1 to 21 days of age, the ADG and ADFI increased while FCR decreased linearly and quadratically (P < 0.0001) as dietary NPP level increased ( Fig. 1a-c) . From 22 to 42 days of age, the ADG and ADFI increased linearly and quadratically (P < 0.0001) as dietary NPP level increased ( Fig. 1e and f) . The mortality of broilers from 1 to 21 or 22 to 42 days of age decreased linearly and quadratically (P < 0.0001) as dietary NPP level increased ( Fig. 1d and h) . Most of the broilers fed the diet with 0.15% dietary NPP died at 22 days of age, and thus we terminated this treatment from d 22 on, and continued the experiment with all of other four treatments.

Tibia BMC, BMD, BBS, and ash concentration Dietary NPP level affected (P < 0.0001) tibia BMC, BMD, BBS and ash concentrations of broilers at 14, 28 and 42 days of age (Fig. 2) . All of the above indices at 14, 28 and 42 days of age increased linearly and quadratically Fig. 1 Effect of dietary NPP level on growth performance and mortality of broilers. A ADG from 1 to 21 days of age. B ADFI from 1 to 21 days of age. C FCR from 1 to 21 days of age. D Mortality from 1 to 21 days of age. E ADG from 22 to 42 days of age. F ADFI from 22 to 42 days of age. G FCR from 1 to 21 days of age. H Mortality from 22 to 42 d of age. All values are expressed as means±SD; Mortality, Percentage data for mortality of birds were transformed to arcsine for analysis; n = 8 for all of other groups except for n = 2 for the first group with 0.15% dietary NPP from d 1 to 21. Lacking the same letters (a, b, c, d) means differences at P < 0.0001. ADG, average daily gain; ADFI, average daily feed intake; feed conversion ratio (FCR); NPP, non-phytate phosphorus (P < 0.05) as dietary NPP level increased (Fig. 2 ). At 14 days of age, all of the above indices increased gradually (P < 0.007) at 0.15-0.45% dietary NPP, and then reached a plateau (P > 0.37) at 0.45-0.55% dietary NPP (Fig. 2a) . At 28 and 42 days of age, all of the above indices increased gradually (P < 0.004) at 0.22-0.36% dietary NPP, and then reached a plateau (P > 0.06) at 0.36-0.43% dietary NPP (Fig.  2b and c).

Dietary NPP level affected serum Ca, phosphorus and 1, 25(OH) 2 D 3 concentrations at 14, 28 and 42 days of age (P < 0.05), and serum PTH concentrations at 28 days of age (P = 0.005), but did not affect serum ALP activity and FGF23 concentrations of broilers at 14, 28 and 42 days of age (P > 0.06), and serum PTH concentrations at 14 and 42 days of age (P > 0.80) (Fig. 3) . At 14 and 42 days of age, serum Ca concentration decreased linearly (P < 0.0004), but at 28 days of age, it decreased linearly (P = 0.05) and quadratically (P = 0.006) as dietary NPP level increased (Fig. 3a, c and e) . At 14 days of age, serum phosphorus and 1, 25(OH) 2 D 3 concentration decreased quadratically (P = 0.02) ( Fig. 3a and b) , however, at 28 and 42 days of age, serum phosphorus increased linearly and quadratically (P < 0.03), and serum 1, 25(OH) 2 D 3 increased linearly (P < 0.03) as dietary NPP level increased (Fig. 3c-f ). At 28 days of age, serum PTH concentrations decreased linearly (P = 0.001) as dietary NPP level increased (Fig. 3d) .

Tibia ash ca and phosphorus concentrations, TP TA , tibia ALP activity and BGP concentration Dietary NPP level affected (P < 0.01) tibia ash phosphorus concentration, TP TA and tibia ALP activity at 14, 28, and 42 days of age, as well as tibia ash Ca and tibia BGP concentrations at 14 days of age, but did not affect (P > 0.17) tibia ash Ca and tibia BGP concentrations on days 28 and 42 ( Fig. 4 and Fig. 5 ). At 14 days of age, tibia ash Ca and TP TA increased linearly and quadratically (P < 0.0001) and tibia BGP concentrations decreased linearly and quadratically (P < 0.008), while tibia ash phosphorus concentration increased linearly (P < 0.0001) and tibia ALP activity decreased linearly (P < 0.0001) as dietary NPP level increased ( Fig. 4a and Fig. 5a) . Tibia ash phosphorus concentration on d 28 increased linearly (P = 0.0009) and on d 42 it increased linearly and quadratically (P < 0.03), while tibia ALP activities on d 28 and 42 decreased linearly and quadratically (P < 0.04) as dietary NPP level increased (Fig. 4b and c) . The TP TA on d 28 and 42 increased linearly and quadratically (P < 0.002) as dietary NPP level increased (Fig. 5b and c) . (Fig. 6) . At 14 and 28 days of age, tibia FGF23 mRNA expression increased linearly or quadratically (P < 0.004), and tibia PHEX mRNA expression decreased linearly (P < 0.04), while tibia DMP1 mRNA expression increased linearly (P < 0.02) as dietary NPP level increased ( Fig. 6a  and b) . Dietary NPP level affected (P < 0.02) tibia DMP1 protein expression at 28 days of age, but did not affect (P > 0.12) tibia FGF23, PHEX, MEPE, and DMP1 protein expressions at 14 and 42 days of age, as well as tibia FGF23, PHEX and MEPE protein expressions of broilers at 28 days of age (Fig. 7 ). At 28 days of age, tibia DMP1 protein expression decreased linearly (P = 0.01) as dietary NPP level increased ( Fig. 7c and d) . Correlations between all of the above bone phosphorus retention or bone development parameters and serum FGF23 on d 14, 28 and 42 were not analyzed because serum FGF23 concentrations at these time points were not affected by dietary NPP level as shown above. Correlations between all of the bone phosphorus retention or bone development parameters and protein expressions of the above local bone-derived regulators or MEPE mRNA and protein expressions on d 14, 28 and 42 were not analyzed because protein expressions of the above local bone-derived regulators or MEPE mRNA and protein expressions at these time points were not affected by dietary NPP level except for the tibia DMP1 protein expression on d 28 as shown above. 

Our hypothesis that bone phosphorus retention and bone development might be regulated not only by the related hormones, but also by the local bone-derived factors in broiler chickens has been supported by the results of the present study. The results from the present study suggested that bone phosphorus retention and bone development might partly be regulated by serum PTH and 1,25(OH) 2 D 3 , tibia FGF23, PHEX, DMP1, ALP and BGP in broiler chickens during the whole growth period. Therefore, these above findings could help us to better understand the mechanisms of bone phosphorus retention and bone development, and provided novel and scientific experimental bases for improving bone phosphorus utilization in broiler chickens.

The results from the present study also indicate that phosphorus plays an important role in promoting the growth, bone development and mineralization in the fast-growing broiler chickens. In the current study, the ADG and ADFI, tibia BMD, BMC, BBS and ash concentrations of broiler chickens were significantly improved by the increase of dietary NPP level in the whole growth period, which is similar to the results reported from our laboratory [21, 35] . Therefore, broiler chickens are very susceptible to dietary phosphorus deficiency, and they could be used as an ideal animal model for studying the bone phosphorus metabolism and bone development and health. However, there were no huge mortality rates for the broilers fed the diets with similar deficient levels of dietary NPP in our previous two studies [35, 36] . The huge mortality rate of broilers fed the diet with 0.15% dietary NPP in the present study might be associated with a very high susceptibility of these birds to dietary phosphorus deficiency. Exact reasons remain to be further studied.

Additionally, many studies have indicated that the ratio of Ca:NPP is more important for growth and bone development of broilers than the absolute concentration of either Ca or NPP [37, 38] . It is reported that a decrease in the dietary levels of Ca and NPP at a constant Ca:NPP ratio (about 2.0:1) improved the FCR, without affecting the growth rate of broilers, but impaired the bone development [39] . Therefore, it needs to be further studied how the results would change if the Ca:NPP ratio had been maintained at a constant ratio (e.g. 1.94:1).

The phosphorus homeostasis is critical for many cellular processes and bone mineralization, which is tightly regulated by several related hormones [40, 41] . The classical calcitropic hormones, such as PTH and 1, 25(OH) 2 D 3 , as well as FGF23, regulates systemic phosphorus levels by creating bone-kidney-parathyroid feedback loops [42] . The PTH is secreted by the parathyroid glands and is the most important regulator of phosphorus levels in the blood and bones [43] . A previous study has shown that a high phosphorus diet increased PTH secretion and then caused an ongoing process in bone reabsorption in rats [44] , and PTH can induce the synthesis of 1,25(OH) 2 D 3 in the kidney, and then in turn, 1,25(OH) 2 D 3 can inhibit PTH secretion by the parathyroid glands [45] . In vehicle-treated hypophosphatemic mice, the serum 1,25(OH) 2 D 3 concentration was inappropriately low for the degree of hypophosphatemia, compared with values in wild type mice [46] , and patients with hypophosphatemia show inappropriately low serum 1,25(OH) 2 D 3 level [47] . In our study, serum 1, 25(OH) 2 D 3 concentrations on d 28 and 42 increased as dietary NPP level increased and had moderate correlations with most of bone phosphorus retention or bone development parameters. Serum PTH concentration on d 28 decreased as dietary NPP level increased and had strong correlations with all of bone phosphorus retention or bone development parameters. The above results from the present study suggested that the two hormones 1,25(OH) 2 D 3 and PTH might regulate bone phosphorus retention and bone development of broilers. However, in the current study, serum FGF23 was not affected by dietary NPP level, which might be due to the concomitant changes in these parameters, such as serum phosphorus and Ca that clearly affected FGF23 levels. It is thus speculated that serum PTH, 1,25(OH) 2 D 3 and FGF23 are controlled within a narrow physiological range by feedback mechanisms and their respective receptors between bone and other organs, and exact mechanisms and their relationships need to be further studied.

The local bone-derived regulators are not only markers of osteocyte maturation, but also functionally involved in phosphorus regulation and bone mineralization [48] . The DMP1 and PHEX are known to be involved in inhibition of FGF23 production [49] . Wang et al. (2018) reported that dietary high phosphorus level up-regulated FGF23 mRNA expression in tibia compared with dietary low phosphorus level in broilers [17] . A study showed that an increased level of inorganic phosphorus in media up-regulated the DMP1 protein expression in primary osteocytes isolated from mouse bones [8] . A study also reported that MEPE mRNA expression was up-regulated in bone and osteoblasts derived from hypophosphatemia mice [50] . The PHEX is the gene whose mutation is the cause of Xlinked hypophosphatemia in humans and mice, and their possible sensitivity to stimulation by low phosphorus diets [51] . In the present study, tibia FGF23 and DMP1 mRNA expressions increased and PHEX mRNA expression decreased on d 14 or 28 as dietary NPP level increased, while tibia MEPE mRNA expression was not affected, but tibia DMP1 protein expression on d 28 decreased as dietary NPP level increased, and protein expressions of DMP1 on d 14 and 42 and the other local bone-derived regulators in tibia of broilers were not affected by dietary NPP. It is noted that protein expressions of tibia FGF23, PHEX and DMP1 were inconsistent with their mRNA expressions on d 14 and 28, which might be due to the effects of some factors, such as translation rate, post-translational modification, greater biological and experimental variations [35] , and [13, 52] . The ALP functions mainly by degrading inorganic pyrophosphate and providing free inorganic phosphorus, and the tissue nonspecific ALP has a positive influence on mineralization primarily by controlling the size of the inhibitory pool of pyrophosphate through its inorganic pyrophosphatase activity [53, 54] . The ALP can be secreted by osteoblasts and regarded as an important component of osteoblastic activity and osteogenesis [55] . Hence, the increase of ALP activity indicated that the bone tissue underwent more intense osteogenesis [56] . However, the increased inorganic phosphorus resulting from the increased ALP activity can be also used for other metabolic functions, such as energy metabolism, cellular signaling, membrane integrity, enzyme activity, and so on [35] . The BGP is a specific product of the osteoblast involved in bone formation and inhibits the hydroxyapatite formation, and thus relatively low concentrations of BGP are present in bone matrix at the onset of mineralization [57, 58] . In our current study, tibia ALP activities on d 14, 28 and 42 decreased as dietary NPP levels increased, which is similar to previous reports from our laboratory [21, 35] , suggesting that birds might obtain phosphorus for important metabolic functions (energy metabolism, cellular signaling, membrane integrity, enzyme activity, etc.) through increasing the bone ALP activity or bone resorption when dietary phosphorus is below the requirement [35] . The above results suggested that bone phosphorus retention and bone development might also be regulated by these local bone-derived regulators (DMP1, PHEX, FGF23, ALP and BGP) of broiler chickens in the whole growth period. To our knowledge, this has been not reported before. However, further studies need be carried out to verify the roles of these local bone-derived regulators in regulating bone phosphorus retention and bone development of broiler chickens.

The results from the present study indicated that adequate dietary phosphorus is essential for enhancing the growth, phosphorus homeostasis in serum or tibia and bone mineralization of fast-growing broiler chickens; and bone phosphorus retention and bone development parameters had moderate to strong correlations with serum PTH and 1,25(OH) 2 D 3 , tibia DMP1, PHEX, FGF23, ALP and BGP in broilers during the whole growth period, and thus they might be partly regulated by these related hormones and local bone-derived regulators. Further studies warrant to be done to verify the roles of these hormones and local bone-derived regulators in regulating bone phosphorus retention and bone development of broiler chickens, and how the results would change if the Ca:NPP ratio had been maintained at a constant ratio (e.g. 1.94:1). 

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Nutrient requirements of poultry

Feeding standard of chicken

An optimal dietary nonphytate phosphorus level of broilers fed a conventional corn-soybean meal diet from 4 to 6 weeks of age

Nonphytate phosphorus requirement and phosphorus excretion of broiler chicks fed diets composed of normal or high available phosphate corn with and without microbial phytase

Relative bioavailability of ultrafine sodium selenite for broilers fed a conventional corn-soybean meal diet

Effect of dietary manganese on antioxidant status and expression levels of heat-shock proteins and factors in tissues of laying broiler breeders under normal and high environmental temperatures

Comparison of the stannous chloride and vanadate methods for estimation of serum inorganic phosphorus by use of the "SMA 12-60

Effect of microbial phytase on ileal digestibility of phytate phosphorus, total phosphorus, and amino acids in a low-phosphorus diet for broilers

Association of Offcial Analytical Chemists. Offcial methods of analysis

Effects of alfalfa and fructooligosaccharide on molting parameters and bone qualities using dual energy X-ray absorptiometry and conventional bone assays

The effects of growth rate on leg morphology and tibia breaking strength, mineral density, mineral content, and bone ash in broilers

Simplified method for the estimation of inorganic phosphorus in body fluids

Effect of manganese source on manganese absorption and expression of related transporters in the small intestine of broilers

Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(−Delta Delta C) method

Principles and procedure of statistics: A biometrical approach

COVID-19 risk assessment tool: dual application of risk communication and risk governance

Dietary non-phytate phosphorus requirement of broilers fed a conventional corn-soybean meal diet from 1 to 21 d of age

Phosphorus absorption and gene expression levels of related transporters in the small intestine of broilers

Nutritional geometry of calcium and phosphorus nutrition in broiler chicks. Growth performance, skeletal health and intake arrays

Dietary calcium or phosphorus deficiency impairs the bone development by regulating related calcium or phosphorus metabolic utilization parameters of broilers

Body weight of young broilers fed with declining calcium and phosphorus contents during the starter period is irresponsive to changes in the skeleton

Molecular interactions of FGF23 and PTH in phosphate regulation

1,25-Dihydroxyvitamin D maintains brush border membrane NaPi2a and attenuates Phosphaturia in Hyp mice

Phosphate as a signaling molecule and its sensing mechanism

Serum levels of phosphorus, parathyroid hormone, and calcium and risks of death and cardiovascular disease in individuals with chronic kidney disease: a systematic review and meta-analysis

High phosphorus diet increases preproPTH mRNA independent of calcium and calcitriol in normal rats

Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia

Chronic inhibition of ERK1/2 signaling improves disordered bone and mineral metabolism in hypophosphatemic (Hyp) mice

Inorganic phosphate homeostasis and the role of dietary phosphorus

MEPE, a new gene expressed in bone marrow and tumors causing osteomalacia

Bone proteins PHEX and DMP1 regulate fibroblastic growth factor Fgf23 expression in osteocytes through a common pathway involving FGF receptor (FGFR) signaling

Mepe, the gene encoding a tumor-secreted protein in oncogenic hypophosphatemic osteomalacia, is expressed in bone

MRNA expression of Phex in mice and rats: the effect of low phosphate diet

Correlations between gene expression and mineralization in the avian leg tendon

Tissue-nonspecific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization

The mechanism of mineralization and the role of alkaline phosphatase in health and disease

Further characterization of ATPinitiated calcification by matrix vesicles isolated from rachitic rat cartilage. Membrane perturbation by detergents and deposition of calcium pyrophosphate by rachitic matrix vesicles

Letrozole inhibits the osteogenesis of medullary bone in prelay pullets

Absence of the vitamin K-dependent bone protein in fetal rat mineral. Evidence for another gammacarboxyglutamic acid-containing component in bone

Origin of the vitamin K-dependent bone protein found in plasma and its clearance by kidney and bone

The authors gratefully acknowledge all professors and students in the mineral nutrition research division.

The data are shown in the main manuscript and supplemental materials.

Ethics approval and consent to participate The experimental protocols used in this experiment, including animal care and use, were reviewed and approved by the Animal Care and Use Ethics Committee of the Institute of Animal Science, Chinese Academy of Agricultural Sciences (Beijing, China) .

Not applicable.

The authors declare that they have no competing interests.