YANG Yun-feng,XlNG Guan-zhong ,Ll Su-fen,SHAO Yu-xinZHANG Li-yangLU LinLUO Xugang,LlAO Xiu-dong

1 Mineral Nutrition Research Division,Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193,P.R.China

2 Poultry Mineral Nutrition Laboratory,College of Animal Science and Technology,Yangzhou University,Yangzhou 225000,P.R.China

3 College of Animal Science and Technology,Hebei Normal University of Science and Technology,Qinhuangdao 066004,P.R.China

Abstract This experiment was conducted to investigate the effect of dietary calcium (Ca) or phosphorus (P) deficiency on bone development and related Ca or P metabolic utilization parameters of broilers from 22 to 42 days of age based on our previous study,which indicated that dietary Ca or P deficiency impaired the bone development by regulating related Ca or P metabolic utilization parameters of broilers from 1 to 21 days of age.A total of 504 one-day-old Arbor Acres male broilers were randomly assigned to 1 of 4 treatments with 7 replicates in a completely randomized design,and fed the normal control and Ca-or P-deficient diets from 1 to 21 days of age.At 22 days of age,the broilers were further fed the normal control diet (0.90% Ca+0.35% non-phytate P (NPP)),the P-deficient diet (0.90% Ca+0.18% NPP),the Ca-deficient diet (0.30%Ca+0.35% NPP) or the Ca and P-deficient diet (0.30% Ca+0.18% NPP),respectively.The results showed that dietary Ca or P deficiency decreased (P＜0.05) tibia bone mineral density (BMD),bone breaking strength (BBS),ash content,tibia ash Ca content and serum P content on days 28 and 42,but increased (P＜0.05) tibia alkaline phosphatase (ALP) activity of broilers on day 42 compared with the control group.Furthermore,the broilers fed the P-deficient diet had the lowest(P＜0.05) tibia BMD,BBS,ash content,serum P content and the highest (P＜0.05) serum Ca content on day 28 compared with those fed the Ca-deficient or Ca and P-deficient diets.The results from the present study indicated that the bone development and related Ca or P metabolic utilization parameters of broilers were the most sensitive to dietary P deficiency,followed by dietary Ca deficiency or Ca and P-deficiency;dietary Ca or P deficiency impaired the bone development possibly by regulating serum Ca and P contents as well as tibia Ca content and ALP activity of broilers from 22 to 42 days of age.

Keywords:calcium deficiency,phosphorus deficiency,bone development,metabolic utilization parameter,broiler

1.lntroduction

Calcium (Ca) and phosphorus (P) are essential minerals involved in many biological process,such as enzyme activation,intracellular signaling,acid-base balance,nucleic acid synthesis and bone mineralization (Berndt and Kumar 2009;Akteret al.2016;Liet al.2017).As most abundant elements in the body,about 99% of Ca and 80% of P were stored in the skeleton as hydroxyapatite (Proszkowiec and Angel 2013).The Ca or P deficiency and improper ratio usually hinder bone growth and lead to a greater incidence of leg abnormality (Valableet al.2017),and if severe enough could cause rickets,especially in broilers during the starter period (Xieet al.2009).It was found that P deficiency could cause poor mineralization of broilers from 1 to 21 days of age,especially impaired the bone mineral density (BMD),bone-breaking strength (BBS) and bone ash content,which were regarded as good indices to reflect overall skeletal health (Bradburyet al.2014;Liuet al.2017).And tibia ash,Ca and P contents of broilers increased curvilinearly with dietary P content increased (Ven?l?inenet al.2006).Additionally,researchers found that low dietary Ca and P not only resulted poorer bone quality but also affected plasma Ca and P contents (Rousseauet al.2012).Alkaline phosphatase (ALP) is a hydrolase involved in the process of Ca and P deposition and closely related to the rate of skeletal mineralization of birds (Tilgaret al.2008).Low dietary Ca affected serum Ca concentration and ALP activity of hens (Jianget al.2013).Vitamin D3can stimulate Ca and P absorption for bone development and modeling,which might play an important role in bone development of poultry(Baret al.1987;Shaoet al.2019a).The 25-hydroxyvitamin D3(25-OHD3) is the active and stable form of vitamin D3,and serum 25-OHD3content is associated with Ca and P metabolic utilization and homeostasis of broilers (Liet al.2020)..Furthermore,the latest study from our laboratory indicated that dietary Ca or P deficiency impaired the bone development possibly by regulating these above related Ca or P metabolic utilization parameters of broilers from 1 to 21 days of age (Liet al.2020).However,few studies have been conducted to investigate the effect of dietary Ca or P deficiency on bone development and related Ca or P metabolic utilization parameters of broilers from 22 to 42 days of age.The Ca-and P-deficient diets increased the broken tibia incidence and decreased the tibia ash percentage of broilers from 1 to 18 and 19 to 35 days of age(Driveret al.2006).It was found that broilers had the ability to partially adapt to Ca and P moderate deficiency from 18 to 32 days of age,which was shown by compensatory growth,and compensatory improvement in bone parameters(Yanet al.2005);but another study demonstrated that low dietary P was harmful to the bone development of broilers from 22 to 42 days of age (Jianget al.2016).Therefore,it is still not clear about dietary Ca or P deficiency on the bone development and related Ca or P metabolic utilization parameters of broilers from 22 to 42 days of age.It was thus hypothesized that the Ca or P metabolic utilization parameters might also play a role in the regulation of bone development of broilers fed the Ca-or P-deficient diets from 22 to 42 days of age.This study was conducted to investigate the effect of dietary Ca or P deficiency on the bone development and related Ca or P metabolic utilization parameters of broilers from 22 to 42 days of age in order to test the above hypothesis.

2.Materials and methods

2.1.Experimental design and treatments

A completely randomized design involving a 2 (dietary Ca levels)×2 (dietary non-phytate P (NPP) levels) factorial arrangement of treatments was used in this experiment.The 2 dietary Ca levels were a normal Ca level of 0.90%and a low Ca level of 0.30%,and the 2 dietary NPP levels were a normal NPP level of 0.35% and a low NPP level of 0.18%.Therefore,there were a total of 4 dietary treatments,including the control diet (0.90% Ca+0.35% NPP),the P-deficient diet (0.90% Ca+0.18% NPP),the Ca-deficient diet (0.30% Ca+0.35% NPP) and the Ca and P-deficient diet (0.30% Ca+0.18% NPP).

2.2.Birds and diets

All experimental procedures were approved by the Animal Management Committee (in charge of animal welfare issue)of the Institute of Animal Sciences,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.A total of 504 one-day-old Arbor Acres male broiler chicks were randomly allotted by body weight (BW) to 1 of 4 treatments with 7 replicate cages of 18 chicks per replicate cage for each treatment.From 1 to 21 days of age,the broilers were fed the normal control and Ca-or P-deficient diets according to our previous study(Shaoet al.2019b).From 22 to 42 days of age,the broilers were further fed the above experimental treatment diets.The basal corn-soybean meal diet (Table 1) was formulated to meet or exceed the nutrient requirements (NRC 1994)for broilers,except for Ca and P.The birds were housed in an electrical heated,thermostatically controlled room with fiberglass feeder (150 cm,length) and strained-steel cages(160 cm×90 cm×75 cm,length×width×height) coated with plastic,and maintained on a continuous 18-h light and 6-h dark of schedule during the experimental period.Feed and tap water were availablead libitum.Dietary treatments were supplemented with Ca and P in the form of CaHPO4,Ca(H2PO4)2and limestone.The diets were fed in the mash form.

2.3.Sample collections and preparations

The feed ingredient and diet samples from all the treatments were taken and submitted for crude protein (CP),Ca and P analyses before the initiation of the trial to confirm CP,Ca and P contents in diets.On days 28 and 42,2 chicks were chosen from each replicate cage according to average BW.Blood samples were taken into tubes without heparinized from each of 2 birdsviawing vein puncture,immediately centrifuged for 10 min at 3 000×g at 4°C for analyzing of serum Ca,P,25-OHD3contents and ALP activity.Then,the selected birds from each replicate were killed by cervical dislocation.The right tibia was peeled and frozen at -20°C for analyses of the BMD,BBS,and the contents of the tibia ash and tibia ash Ca and P.The left tibia was peeled and frozen at -20°C for analysis of the ALP activity.Samples from 2 chicks in each replicate cage were pooled into 1 sample in equal ratio before analysis.

2.4.Determinations of feed ingredients and diets

The concentrations of CP and P in feed ingredient and diet samples were determined as described by AOAC(2000).The concentrations of Ca in feed ingredient and diet samples were measured by inductively coupled plasma emission spectroscope (Model IRIS Intrepid II,Thermal Jarrell Ash,Waltham,MA,USA) after wet digestions with HNO3and HClO4as described by Luoet al.(2005).The phytate phosphorus (PP) contents in diets were analyzed according to the ferric precipitation method described by Leytemet al.(2008).Therefore,dietary NPP was calculatedas the difference between total P and PP.

Table 1 Composition and nutrient levels of experimental diets (as-fed basis)

2.5.Determinations of the serum Ca,P,25-OHD3 contents as well as serum and tibia ALP activity

Serum was thawed and analyzed for the Ca content using a microplate reader with Ca assay kits (Nanjing Jiancheng Bioengineering Institute,Nanjing,China).Serum P content was determined by molybdenum blue method according to Goldenberg and Fernandez (1966).The 25-OHD3level in serum was determined by the method of ELISA with 25-Hydroxyvitamin D Assay Kits (Nanjing Jiancheng Bioengineering Institute,Nanjing,China).The ALP activity in serum and tibia were measured using a microplate reader with ALP Assay Kits (Nanjing Jiancheng Bioengineering Institute,Nanjing,China).

2.6.Determinations of the tibia BMD,BBS,ash content and ash Ca and P contents

The frozen tibia was thawed at room temperature for 2 h,defleshed and then stripped of all soft tissues.The BMD were determined by dual-energy X-ray absorptiometry(DEXA) using the case of small animals model (DCS-600;Aloka,Tokyo,Japan) as describes by Liuet al.(2017).After the scan,tibia bones were immediately rebagged and frozen until analysis of bone strength (Jianget al.2016).The tibia bone strength was determined by a three-point bending test (Electronic Universal Testing Machine Wd-1;Changchun Non-Metallic Materials Testing Machine Factory,Changchun,China).The tibia bone was put on a fulcrum point with 70 mm apart.Loading point was located in the midpoint of fulcrum points.The value of breaking force was determined by the shear test at a speed of 5 mm min-1with a 50-kg loading cell until fracture occurred (Crenshawet al.1981).The ultimate breaking force of the tibia bone was indirectly obtained,according to the loaded-deformation curve recorded by computer.The tibia bone was dried in an oven at 105°C for 24 h and then defatted with fresh diethyl ether for 48 h.The fat-free,dried bone was finally ashed in a muffle furnace at 550°C for 16 h.The tibia ash contents were expressed on a dried and defatted weight basis of tibia.The Ca content in tibia ash was determined by inductively coupled plasma spectroscopy (Model IRIS Intrepid II;Thermo Jarrell Ash,Waltham,MA,USA).Total P content in tibia ash was determined with a spectrophotometer(AOAC 2000).

2.7.Statistical analyses

Data on day 28 or on day 42 from the present study were subjected to two-way or one-way ANOVA using the general linear model (GLM) procedure of SAS 9.4 (SAS Institute Inc.,Cary,NC),respectively.The model for two-way ANOVA included the main effects of dietary Ca level,dietary P level and their interaction.One replicate is regarded as an experimental unit.Differences among means were tested by the least significant difference (LSD) method,and the statistical significance was set atP＜0.05.

3.Results

3.1.Tibia BMD,BBS and ash content

Dietary Ca level,NPP level and their interaction affected(P＜0.05) BMD,BBS and ash content in tibia of broilers on day 28,and dietary Ca or P deficiency also affected(P＜0.05) these indices of broilers on day 42 (Table 2).The broilers fed the control diet had higher(P＜0.05) tibia BMD,BBS and ash content than those fed the Ca-or P-deficient diets,furthermore,the broilers fed the P-deficient diet had the lowest tibia BMD,BBS and ash content than those fed the other diets on day 28.However,no differences (P＞0.05)were observed in these indices between the Ca-deficient group and the Ca and P-deficient group.The mortality of the experimental broilers from 1 to 21 days of age were found to be 5.56,26.19,0.79,and 1.59% in control group,P-deficient group,Ca-deficient group and Ca and P-deficient group,respectively.All the broilers in the P-deficient group died at 29 days of age.The mortality of control group,Cadeficient group and Ca and P-deficient group from 22 to 42 days of age were 6.35,3.17,and 0%,respectively.

3.2.Serum Ca,P,25-OHD3 contents and ALP activity

Dietary Ca level did not affect (P＞0.05) Ca,P and 25-OHD3contents and ALP activity in serum of broilers on day 28(Table 3).Dietary NPP level and the interaction between Ca and NPP level had effects (P＜0.05) on serum Ca,P and 25-OHD3contents,but had no effects (P＞0.05) on ALP activity on day 28.Dietary Ca or P deficiency did not affect (P＞0.05)serum Ca content,ALP activity and 25-OHD3content,but affected (P＜0.05) serum P content of broilers on day 42.The broilers fed P-deficient diet had higher (P＜0.05) Ca content and lower (P＜0.05) P content in serum than those fed the control diet,but the broilers fed Ca and P-deficient diet had lower (P＜0.05) Ca content and higher (P＜0.05) P content in serum than those fed the Ca-deficient diet.No difference(P＞0.05) was observed in serum 25-OHD3content on day 28 between the broilers fed the control and P-deficient diets;however,the broilers fed Ca and P-deficient diet had lower(P＜0.05) serum 25-OHD3content on day 28 than those fed the Ca-deficient diet.The broilers fed the control diet had higher (P＜0.05) serum P content on day 42 than those fed Ca-deficient and Ca and P-deficient diets.

3.3.Tibia ash Ca,P contents and ALP activity

Dietary Ca level had effect (P＜0.05) on tibia ash Ca,P contents and ALP activity on day 28(Table 4).Dietary NPP level affected (P＜0.05)tibia ash Ca content and ALP activity,but did not affect (P＞0.05)tibia ash P content on day 28.The interaction between Ca and NPP levels affected(P＜0.05) tibia ash Ca content,but did not affect (P＞0.05) tibia ash P content and ALP activity on day 28.Dietary Ca or P deficiency affected(P＜0.05) tibia ash Ca content and ALP activity,but did not affect (P＞0.05) tibia ash P content of broilers on day 42.The broilers fed control diet had higher (P＜0.05) tibia ash Ca content than those fed P-deficient diet;however,no difference (P＞0.05)was observed in tibia ash Ca content on day 28 between the broilers fed the Cadeficient and Ca and P-deficient diets.Compared with the broilers fed diet with 0.90% Ca,the broilers fed diet with 0.30% Ca had higher (P＜0.05)tibia ash P content and ALP activity on day 28.Broilers fed diet with 0.18% NPP had higher (P＜0.05) tibia ALP activity than those fed diet with 0.35% NPP on day 28.The broilers fed control diet had higher (P＜0.05) tibia ash Ca content and lower (P＜0.05) tibia ALP activity than those fed the Ca-deficient diet or the Ca and P-deficient diet.

4.Discussion

The hypothesis that the Ca or P metabolic utilization parameters might also play a role in the regulation of bone development of broilers fed the Ca-or P-deficient diets from 22 to 42 days of age was supported by the results of the present study.The present study demonstrated that dietary Ca or P deficiency impaired the bone development by regulating serum Ca and P contents as well as tibia Ca content and ALP activity of broilers from 22 to 42 days of age.The above and our previous findings provided scientific experimental bases for monitoring and regulating Ca and P homeostasis of bones in broilers for the whole growth periods.

Many studies have demonstrated that dietary Ca or P play a critical role in the bone development,such as tibia BMD,BBS and ash content of broilers(Williamset al.2000;Raoet al.2003;Liuet al.2017).Results from the current study also showed that dietary Ca or P deficiency negatively affected the tibia BMD,BBS and ash content of broilers from 22 to 42 days of age.Similar reports showed that broilers fed diet with 0.19% NPP had lower tibia BMD,BBS and ash content than those fed diet with 0.34% NPP from 22 to 42 days of age (Jianget al.2016).Bone strength was also found higher for birds fed the high-Ca diet than those fed the low-Ca diet (Christensenet al.2003).Moreover,the present study showed that these tibia development parameters of broilers were the most sensitive to the P-deficient diet,followed by the Ca-deficient diet or Ca and P-deficient diet.Many researchers reported that reduction in dietary P could be achieved without deleterious effects on bone mineralization if Ca is reduced concomitantly,and the reason might be that the Ca:P ratio is still within the range between 2:1 and 1:1,which is generally acceptable for poultry industry (Driveret al.2005;Raoet al.2006).It has been reported that high dietary level of Ca could be tolerated by chickens without any significant effect when providing the corresponding high level of P (Shafey 1993).The tibia component of broilers was directly correlated with the Ca:P ratio of the diet,not the absolute dietary level of either Ca or P (Dilworth and Day 1965).Thus,the most serious depression in bone development parameters of P deficiency group may be due to the improper ratio of Ca:P.A research suggested that relative high Ca reduced P availability (Qianet al.1996),which may be the formation of extremely insoluble Ca-phytate complexes,resulting in a severe deficiency of P,and increased dietary Ca increased intestinal pH,which reduced the soluble fraction of minerals and limited the availability for absorption.

The Ca and P contents in serum and bone can well reflect the nutritional status of Ca and P in the body of broilers.As we expected,dietary Ca or P deficiency decreased Ca content in tibia of broilers,which was similar to the results of Liet al.(2012) to some extent,who found that tibia Ca content was decreased when broilers fed diet with low Ca.The content of tibia P was not affected by NPP,which was partly consistent with the results of Akteret al.(2016),who reported that dietary NPP had no effect on tibia P content.In our current study,the broilers fed 0.18% NPP diet had severely lower serum P level and higher serum Ca level compared with the broilers fed 0.35%NPP diet.Similar results were also reported by Viveroset al.(2002).The low-P diet caused an elevated ionized Ca in the plasma and depressed the release of parathyroid hormone,thus reducing inhibition of parathyroid hormone on tubular re-absorption of phosphate and permitting urinary excretion of additional Ca absorbed from the gut during feeding of a low-P diet (Tayloret al.1968).At the same time,bone resorption was induced to maintain a normal plasma P concentration,and simultaneously increasing serum Ca level (Proszkowiec and Angel 2013).These findings suggested that dietary Ca or P deficiency might impair the bone development by disturbing Ca and P homoeostasis of broilers.

Dietary supplemental vitamin D3could improve the bone mineralization of broilers by increasing the tibia weight,length,width,ash and tibia P retention rate (Hanet al.2015;Shaoet al.2019a),and supplemental 1α-OHD3in low-Ca and low-P diets could compensate the reduction of tibia strength as a result of feeding with deficient diets (Ebrahimiet al.2016).As a metabolite of vitamin D3,25-OHD3could transform to 1,25-dihydroxy-vitamin D3to promote bone mineralization.Chicks are able to adapt partially to a moderate deficiency Ca and P through a compensatory elevation of 1,25-dihydroxy-vitamin D3(Blahoset al.1987),which was consistent with our results that the broilers fed Ca-deficient diet had higher serum 25-OHD3content than other treatment groups.The ALP can be used as a general indicator of skeletal development in vertebrate animals and bone ALP activity predicted the rate of mineralization of leg(Tilgaret al.2008).The present study showed that Ca or P deficiency elevated tibia ALP activity,but had no effect in serum ALP activity.However,serum and tibia ALP activity of broilers were found increased linearly as dietary NPP level decreased on day 21 (Liuet al.2016).The disparity in serum ALP activity may be due to nearly half of serum ALP originated from liver.Similar results showed that increased ALP activity resulting from a dietary Ca deficiency (Hurwitz and Griminger 1961).It was found that the ALP elavated in cases of improper calcification or in diseases involving decalcification of bone such as osteoporosis (Kay 1935;Motzoket al.1950).Therefore,the above results indicated that dietary Ca or P deficiency might impair the bone development by up-regulating tibia ALP activity of broilers.

5.Conclusion

The results from the current study indicated that the bone development and related Ca or P metabolic utilization parameters of broilers were the most sensitive to dietary P deficiency,followed by dietary Ca deficiency or Ca and P-deficiency;dietary Ca or P deficiency impaired the bone development possibly by regulating serum Ca and P contents as well as tibia Ca content and ALP activity of broilers from 22 to 42 days of age.

Acknowledgements

The present study was financially supported by the National Key R&D Program of China (2017YF0502200),the Key Program of the National Natural Science Foundation of China (31630073), the earmarked fund for China Agriculture Research System (CARS-41),the Agricultural Science and Technology Innovation Program,China (ASTIP-IAS09),and the earmarked fund for Hebei Chicken Innovation Team of Modern Agro-Industry Technology Research System,China(HBCT2018150203 and HBCT2018150206).