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Dietary calcium requirements of broilers fed a conventional corn-soybean meal diet from 1 to 21 days of age

Dietary calcium requirements of broilers fed a conventional corn-soybean meal diet from 1 to 21... Background: The current calcium (Ca) recommendation for broilers is primarily based on studies conducted more than 30 years ago with birds of markedly different productive potentials from those which exist today. And the response indicators in these studies are mainly growth performance and bone ash percentage. Therefore, the present study was carried out to investigate the effect of dietary Ca level on growth performance, serum parameters, bone characteristics and Ca metabolism-related gene expressions, so as to estimate dietary Ca requirements of broilers fed a conventional corn-soybean meal diet from 1 to 21 days of age. Methods: A total of 420 1-day-old Arbor Acres male broilers were randomly assigned to 1 of 7 treatments with 6 replicates (10 birds per cage) and fed the corn-soybean meal diets containing 0.60%, 0.70%, 0.80%, 0.90%, 1.00%, 1.10% or 1.20% Ca for 21 days. Each diet contained the constant non-phytate phosphorus content of about 0.39%. Results: The average daily gain decreased linearly (P < 0.001) as dietary Ca level increased. The serum and tibia alkaline phosphatase (ALP) activities, tibia bone mineral density (BMD), middle toe BMD, tibia ash percentage, tibia breaking strength, and tibia ALP protein expression level were affected (P < 0.05) by dietary Ca level, and showed significant quadratic responses (P < 0.02) to dietary Ca levels. The estimates of dietary Ca requirements were 0.80 to 1.00% based on the best fitted broken-line or quadratic models (P < 0.03) of the above serum and bone parameters, respectively. Conclusions: The results from the present study indicate that the Ca requirements would be about 0.60% to obtain the best growth rate, and 1.00% to meet all of the Ca metabolisms and bone development of broilers fed a conventional corn-soybean meal diet from 1 to 21 days of age. Keywords: Bone characteristic, Broiler, Calcium, Gene expression, Requirement * Correspondence: wlysz@263.net; lulin1225@163.com Shiping Bai and Yunfeng Yang contributed equally to this work. Poultry Mineral Nutrition Laboratory, College of Animal Science and Technology, Yangzhou University, Yangzhou 225000, China Mineral Nutrition Research Division, State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China Full list of author information is available at the end of the article © The Author(s). 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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Journal of Animal Science and Biotechnology (2022) 13:11 Page 2 of 12 Background and bone morphogenetic protein-2 (BMP-2) were con- Calcium (Ca) is an essential mineral element, and sidered as good biological markers of bone metabolisms plays an important role in many biological processes, [16–19]. However, the above mentioned indicators have such as enzyme activation, intracellular signaling, never been used to estimate dietary Ca requirements of acid-base balance and bone mineralization [1]. As chickens. We hypothesized that BMD, BMC and mRNA about 99% of Ca is stored in skeleton as hydroxyapa- or protein expression levels of ALP, OC, OPG and tite, Ca is crucial to the bone development of broilers BMP-2 in bone might be new sensitive indices to evalu- [2]. Skeletal abnormalities attract great concern in ate dietary Ca requirements of broilers fed a corn- poultry industryastheycause notonlyproduction soybean meal diet, and dietary Ca requirements of problems but also considerable animal welfare issues. broilers from 1 to 21 days of age might be different from The deficiencies in content or improper ratios of Ca the current NRC Ca requirement (1.00%). and phosphorus (P) were usually considered as main Therefore, the objective of the current study was to in- nutritional reasons leading to greater incidences of leg vestigate the impact of dietary Ca levels on growth per- abnormalities and huge economic loss [3]. formance, serum parameters, bone characteristics and The current Ca recommendations for broilers by Ca metabolism-related gene expressions of broilers, so NRC (1994) [4] were 1.00%, 0.90%, and 0.80% for the as to choose sensitive indices to evaluate dietary Ca re- starter (1 to 21 d), grower (22 to 42 d), and finisher (43 quirements of broilers fed a corn-soybean meal diet to 56 d) phases, respectively. However, these recommen- from 1 to 21 days of age. dations are based on studies conducted in 1960s and 1980s [5, 6], and the response indicators in these studies Materials and methods are mainly growth performance and bone ash percent- All experimental procedures were approved by the Ani- age. The modern broilers differ very much from those mal Management Committee of the Institute of Animal before 1980s in the growth rate, feed conversion effi- Science, Chinese Academy of Agricultural Sciences ciency, carcass quality and bone characteristics [7–9]. (IAS-CAAS, Beijing, China), and performed in accord- The feed efficiency of modern broiler chickens has been ance with the guidelines. Ethical approval on animal sur- improved [7], and it is assumed that the feed intake has vival was given by the animal ethics committee of IAS- decreased, thus the modern broiler chickens have pro- CAAS. The ARRIVE guidelines were followed for report- duced the same adequate bone traits with reduced Ca. ing animal research [20]. Therefore, the Ca requirements of modern fast-growing broilers might be different from those of broiler strains Experimental design, animals and diets reared several decades ago. However, as Ca appears to A total of 420-day-old Arbor Acres male broiler chicks be less threatening to the environment than P [10], and (Huadu Broiler Breeding Corp) with similar body the sources of Ca, namely limestone or oyster shell flour, weights were randomly allotted to 1 of 7 treatments with are cheaper than other mineral sources, the determin- 6 replicate cages of 10 birds per cage in a completely ation of Ca requirements receives little attention [11]. It randomized design, and housed in an electrically heated, has also been reported that modern broilers have a high thermostatically controlled room with fibreglass feeders, capacity to adapt P or Ca deficiency [12]. Nevertheless, waterers and stainless-steel cages coated with plastics for the high level of dietary Ca may reduce the energy value 21 d. They were maintained on a 24-h constant light of the diet and interferes with the availability of other schedule and allowed ad libitum access to experimental minerals [13]. Decreasing the amount of dietary Ca may diets and tap water. The basal corn-soybean meal diet improve performance, but this should not be at the ex- (Table 1) was formulated to meet or exceed the require- pense of increased leg problems [14]. Furthermore, the ments [4, 21] of broilers for all other nutrients except starter phase (1 to 21 d) is a critical period for bone de- for Ca and P. Dietary Ca levels for 7 treatments were velopment of broilers. Thus it is necessary to re-evaluate calculated to be 0.60%, 0.70%, 0.80%, 0.90%, 1.00%, dietary Ca requirements of broilers fed a conventional 1.10% and 1.20% Ca, respectively. The requirement value corn-soybean meal diet from 1 to 21 days of age. of dietary non-phytate P (NPP) recommended by the Chicken bone mineral density (BMD) or content current NRC [4] is 0.45%. In the present study, each diet (BMC) determined by dual-emission X-ray absorpti- contained the constant NPP content of about 0.39% ometry (DEXA) was shown to be highly correlated with based on the results of our previous study [22]. A single bone ash percentage, which could well reflect the Ca nu- large batch of the basal diet without limestone, was tritional status [15]. Furthermore, the enzymes and pro- mixed at first, and then divided into 7 sublots according teins involved in Ca utilization were found sensitive to to the experimental treatment. Each sublot was mixed the dietary Ca levels in animals, especially alkaline phos- with limestone (containing 38.7% Ca by analysis) or fine phatase (ALP), osteocalcin (OC), osteoprotegerin (OPG), sand [23, 24]. The washed fine sand containing no Bai et al. Journal of Animal Science and Biotechnology (2022) 13:11 Page 3 of 12 Table.1 Composition and nutrient levels of the basal diet for collected and stored at − 20 °C for analyses of bone char- broilers from 1 to 21 days of age (as-fed basis) acteristics. The right tibia was snap-frozen in liquid N Item Contents and then stored at − 80 °C for assays of ALP, BMP-2, OC or OPG mRNA and protein expression levels. To re- Ingredients, % duce individual biological variation, samples from two Corn 52.97 chickens in each replicate cage were pooled into one Soybean meal 37.51 sample in equal ratios before analyses, and thus there Soybean oil 5.00 were a total of 6 replicate samples for each treatment. CaHPO 1.72 Limestone 0.24 Serum parameters Salt 0.30 Serum inorganic P was determined by vanadate- DL-Met 0.31 molybdate method [25]. Serum Ca and ALP activities in serum and tibia were measured using commercial assay Micronutrents 0.33 kits (Nanjing Jiancheng Bioengineering Institute, Nan- Sand 1.62 jing, China). Nutrient levels, % ME, MJ/kg 12.69 Bone characteristics Crude protein 21.62 The frozen tibia and middle toe were thawed at room Lysine 1.12 temperature for 2 h, and BMC and BMD were deter- Methionine 0.59 mined by DEXA using the case of small animal model Threonine 0.80 (Lunar_iDXA; GE healthcare, Madison, WI, USA). After the scan, tibia and middle toe were immediately Tryptophane 0.23 rebagged and frozen until further analyses. The tibia Methionine + Cystine 0.90 bone strength was determined using a texture analyser Non-phytate phosphorus 0.39 (TA.XT plus; Stable Micro Systems, London, UK). The Calcium 0.59 tibia was put on a fulcrum point with 50 mm apart. Feed grade Loading point was located in the midpoint of fulcrum Provided per kilogram of diet: VA, 15,000 IU; VD , 4500 IU; VE, 24 IU; VK ,3 3 3 points. The value of breaking force was determined by mg; VB , 3 mg; VB , 9.6 mg; VB , 3 mg;VB , 0.018 mg; Pantothenic acid 1 2 6 12 calcium, 15 mg; Niacin, 39 mg; Folic acid, 1.5 mg; Biotin, 0.15 mg; Choline, 700 the shear test at a speed of 5 mm/min with a 50 kg load- mg; Zn (ZnSO .7H O) 60 mg; Cu (CuSO � 5H O) 8 mg; Mn (MnSO � H O) 110 mg; 4 2 4 2 4 2 ing cell until fracture occurred. The ultimate breaking Fe (FeSO � 7H O) 40 mg; I (KI) 0.35 mg; Se((Na SeO ) 0.35 mg 4 2 2 3 Determined values based on triplicate measurements, and the others were force of the tibia was indirectly obtained, according to calculated values the load v. deformation curve recorded by computer. Following this testing, the tibia and middle toe were detectable Ca and P was used to maintain the same dried at 105 °C for 24 h and defatted with fresh diethyl weight of each treatment diet. The dietary Ca levels by ether for 48 h, and then dried at 105 °C for 12 h to deter- analysis on an as-fed basis were 0.59%, 0.70%, 0.80%, mine bone weight. The dried and defatted tibia and mid- 0.88%, 0.98%, 1.08% and 1.18%, respectively. All diets dle toe were ashed in muffle furnace at 550 °C for 24 h were fed in mash form. At the end of the experiment, to measure bone ash weight and calculate bone ash after fasting for 12-h, broiler weight and feed intake were percentage. recorded for each replicate cage and corrected for mor- tality to calculate average daily gain (ADG), average daily feed intake (ADFI) and feed: gain (FCR) from 1 to 21 Diet and bone ash analyses days of age. Samples of diets and bone ash were ground in a labora- tory mill to pass through a 0.5 mm screen. The crude Sample collections and preparations protein level in the basal diet was analyzed according to At the end of the experiment, chicks in each cage were the Kjeldahl method [26]. The Ca concentrations in diets individually weighed after fasting for 12-h, and 12 chicks and bone ash were determined by inductively coupled (2 chicks per cage) from each treatment were selected plasma spectroscopy (Model IRIS Intrepid II; Thermo according to average body weight of each cage, respect- Jarrell Ash, Waltham, MA, USA). Total P concentrations ively. Blood samples were promptly obtained using wing in diets and bone ash were determined with a spectro- vein puncture, and then centrifuged to harvest serum photometer [27]. Validations of Ca and total P analyses and stored at − 20 °C for analyses of ALP activity and Ca were conducted concurrently using soybean powder or inorganic P contents. Birds were then killed by cer- (GBW10013; National Institute of Standards and Tech- vical dislocation. The left tibia and middle toe were nology, Beijing, China) as a standard reference material. Bai et al. Journal of Animal Science and Biotechnology (2022) 13:11 Page 4 of 12 RNA extraction and quantitative RT-PCR assays gel, and then electrotransferred onto the polyvinylidene The total RNA was isolated from the tibia by using TRI- fluoride membranes (Merck-Millipore, Munich, ZOL reagent (Invitrogen Life Technologies, Carlsbad, Germany). After the transfer, membranes were blocked CA, USA) according to the manufacturer’s instructions. for 1 h at room temperature in a blocking buffer with The concentration of total RNA was estimated by meas- 5% non-fat milk, and then incubated overnight at 4 °C uring its optical density at 260 and 280 nm with a spec- with the following primary antibodies: ALP (A6866; trophotometer (ND-100; NanoDrop Technologies, ABclonal, Wuhan, China), OPG (A135204; ABclonal, Wilmington, DE, USA). A 500 ng of total RNA was re- Wuhan, China), OC (A18241; ABclonal, Wuhan, China), versely transcribed into cDNA using PrimeScript™ RT BMP-2 (A0231; ABclonal, Wuhan, China) and β-tubulin Master Mix Kit (TaKaRa Bio Inc., Otsu, Japan) accord- (HX1829; Huaxingbio, Beijing, China). After washing, ing to the manufacturer’s instructions. The cDNA was the membranes were incubated with the secondary anti- used as templates for real-time quantitative PCR amplifi- body of goat anti-rabbit (HX2028; Huaxingbio, Beijing, cation using SYBR green master mix (Applied Biosys- China) or goat anti-mouse (HX2113; Huaxingbio, tems, Foster City, CA, USA) on the ABI 7500 Real-Time Beijing, China) for 1 h at room temperature. The signals PCR machine following the manufacturer’s guidelines. were recorded with automatic chemiluminescence im- The gene-specific primers for ALP, BMP-2, OPG, OC, β- aging analyzer (5200 Multi; Tanon, Shanghai, China) by actin and glyceraldehyde-3-phosphate dehydrogenase Chemistar ECL Western Blotting Substrate (180–501; (GAPDH) are shown in Table 2. Internal reference Tanon, Shanghai, China). Data were presented as the ra- genes, both β-actin and GAPDH, were constant across tio of ALP, OPG, OC or BMP-2 protein band intensity the dietary treatment groups, and thus their geometric to β-tubulin protein band intensity. mean could be used to normalize the expression of the targeted gene [28]. Relative gene expression was calcu- Statistical analyses 2-ΔΔCt lated using the method [29]. Data from the present study were subjected to one-way ANOVA using the general linear model procedure of Western blotting assays SAS (v. 9.2, SAS Inst. Inc., Cary, NC, USA), and differ- After ground in liquid N , the tibia samples were ho- ences among means were tested by the least significant mogenized in 0.5 mL of ice-cold radio immunoprecipita- difference method. The replicate cage of 9 to 10 chick- tion assay lysis buffer (Beyotime Biotechnology, Haimen, ens for growth performance or two chickens for other China) supplemented with 5 μL of protease inhibitor indicators served as the experimental unit for all statis- (BioTool, Houston, TX, USA). The homogenate was tical analyses. Data from mortality were transformed to centrifuged at 10,000 × g for 10 min at 4 °C, and the arcsine for analysis. Orthogonal comparisons were ap- supernatant was collected for total protein determin- plied for linear and quadratic responses of dependent ation using a BCA Protein Assay Kit (Beyotime Biotech- variables to independent variables. Regression analyses nology, Shanghai, China). The extracted protein (20 μg) of broken-line, quadratic and asymptotic models were was subjected to electrophoresis on a 10% SDS-PAGE performed, and the best fitted models between Table.2 Primer sequences for real-time PCR amplification Gene GenBank ID Primer sequences Length, pb β-actin NM_205518.1 F: 5′-ACCTGAGCGCAAGTACTCTGTCT-3′ 152 R: 5′-CATCGTACTCCTGCTTGCTGAT-3′ GAPDH K01458 F: 5′-CTTTGGCATTGTGGAGGGTC-3′ 128 R: 5′-ACGCTGGGATGATGTTCTGG-3′ ALP NM_205360.1 F: 5′-GGAGAAGGACCCCGAATACTG-3′ 300 R: 5′-TTGACGCCGCAGAGGTAAG-3′ OPG XM_015283019.2 F: 5′-ATCTCAGTCAAGTGGAGCATC-3′ 186 R: 5′-GTTCCAGTCTTCAGCGTAGTA-3′ OC NM_205387.3 F: 5′-TGCTCGCAGTGCTAAAGCCTTCAT-3′ 143 R: 5′-TCAGCTCACACACCTCTCGTT-3′ BMP-2 NM_204358.1 F: 5′-CCAACACCGTGTGCAGCTT-3′ 136 R: 5′-TGGAGTTCAGCTGAGGTGACAGA-3′ Abbreviations: GAPDH glyceraldehyde-3-phosphate dehydrogenase, ALP alkaline phosphatase, OPG osteoprotegerin, OC osteocalcin, BMP-2 bone morphogenetic protein-2 Bai et al. Journal of Animal Science and Biotechnology (2022) 13:11 Page 5 of 12 responsive criteria and dietary Ca level concentrations but tibia BMC increased linearly (P = 0.012). In addition, were used to determine dietary Ca requirements (the tibia breaking strength increased linearly (P = 0.001) and break point from broken-line model or the maximum quadratically (P = 0.012) with increasing dietary Ca response from quadratic model) of broilers [30]. The levels. Tibia ash percentage or breaking strength reached level of statistical significance was set at P ≤ 0.05. a plateau, and tibia BMD reached the highest point at 0.88% Ca. Results Growth performance and mortality Middle toe characteristics Dietary Ca level affected (P< 0.005) body weight on d Dietary Ca level did not influence (P > 0.11) the middle 21 and ADG of broilers from 1 to 21 days of age, but toe ash percentage, P content, Ca to P ratio and BMC, did not affect (P> 0.10) ADFI, FCR and mortality but influenced (P < 0.009) its ash Ca content and BMD (Table 3). The body weight on d 21 and ADG decreased (Table 6). The middle toe ash Ca content increased linearly (P < 0.0001) as dietary Ca level increased. linearly (P < 0.0001), but its BMD increased quadratically Broilers had the highest ADG at dietary Ca level of (P < 0.0001) as dietary Ca level increased. The highest 0.59%. Furthermore, all broilers did not show leg abnor- middle toe BMD was observed visually at 0.88% dietary mality during the whole experimental period. Ca level. Serum parameters and ALP activity in the tibia mRNA levels of ca metabolism-related genes in the tibia Dietary Ca level did not affect (P > 0.05) serum Ca and Dietary Ca level affected (P < 0.03) tibia OC and BMP-2 inorganic P contents, and Ca to inorganic P ratio of mRNA expression levels, but did not affect (P > 0.30) broilers (Table 4), but affected (P < 0.05) ALP activities tibia ALP and OPG mRNA expression levels (Table 7). in serum and tibia. As dietary Ca level increased, the As dietary Ca level increased, both OC and BMP-2 ALP activity in serum increased linearly (P= 0.0003) and mRNA levels decreased linearly (P < 0.03). quadratically (P = 0.002), and it in tibia decreased qua- dratically (P = 0.005). The minimum ALP activity in serum was observed at 0.98% Ca level, and that in tibia Protein expression levels of ca metabolism-related genes at 0.80% Ca level. in the tibia Dietary Ca level affected (P = 0.04) tibia ALP protein ex- Tibia characteristics pression levels, but did not affect (P > 0.24) tibia OPG, Dietary Ca level did not affect (P > 0.39) tibia ash Ca or OC and BMP-2 protein expression levels (Table 7). The P contents and Ca to P ratio, but affected (P < 0.03) the tibia ALP protein expression level increased quadrati- tibia ash percentage, BMD, BMC and breaking strength cally (P = 0.006) as dietary Ca level increased. The high- (Table 5). As dietary Ca level increased, tibia ash per- est tibia ALP protein expression level was observed centage and BMD increased quadratically (P < 0.004), visually at 0.88% dietary Ca level. Table.3 Effect of dietary calcium (Ca) level on the growth performance of broilers during 1 to 21 d of age Analyzed dietary Ca level, % Body weight on d 21, g ADFI, g/d ADG, g/d FCR, g/g Mortality ,% a a 0.59 825 46.32 37.13 1.25 1.67 b b 0.70 793 44.90 35.64 1.26 1.67 bc bc 0.80 781 44.43 35.09 1.27 3.33 b b 0.88 792 44.98 35.58 1.26 0.00 b bc 0.98 789 44.83 35.46 1.27 0.00 bc bc 1.09 768 43.83 34.46 1.27 0.00 c c 1.18 757 43.43 33.94 1.28 0.00 SEM 10.8 1.668 0.096 0.029 0.017 P-value Ca level 0.004 0.11 0.004 0.71 0.33 Linear < 0.0001 – < 0.0001 –– Quadratic 0.72 – 0.72 –– Data represented the means of 6 replicates (n =6) Mortality was based on analysis after anti-sine transform a-c Means within a column with unlike superscript letters were significantly different (P < 0.05) Bai et al. Journal of Animal Science and Biotechnology (2022) 13:11 Page 6 of 12 Table.4 Effect of dietary calcium (Ca) level on serum parameters and tibia ALP activities of broilers on d 21 Analyzed dietary Serum Ca content, Serum inorganic P, Serum Ca: Serum ALP Activity, King Tibia ALP Activity, King Ca level, % mg/100 mL mg/100 mL inorganic P ratio unit/100 mL unit/g prot cd a 0.59 8.63 6.22 1.39 312 384 bc ab 0.70 8.65 7.09 1.23 380 350 cd c 0.80 8.21 6.78 1.22 304 271 bcd bc 0.88 8.50 7.14 1.19 345 305 d ab 0.98 8.95 6.92 1.30 294 340 b ab 1.09 8.63 6.81 1.27 396 348 a ab 1.18 8.76 6.36 1.39 486 355 SEM 0.553 0.534 0.133 51.9 57.1 P-value Ca level 0.43 0.06 0.08 < 0.0001 0.04 Linear –– – 0.0003 0.82 Quadratic –– – 0.002 0.005 Data represented the means of 6 replicates (n =6) Abbreviation: ALP alkaline phosphatase a-d Means within a column with unlike superscript letters were significantly different (P < 0.05) Estimations of dietary ca requirements of broilers the Ca requirement would be about 0.60% to obtain the Results of dietary Ca requirements of broilers as esti- best growth rate and 1.00% to meet all of the Ca metab- mated by the non-linear regression analyses are shown olisms and bone development of broilers fed a conven- in Table 8. The results indicate that serum and tibia tional corn-soybean meal diet from 1 to 21 days of age. ALP activities, tibia ash percentage, tibia breaking strength, tibia and middle toe BMD, and tibia ALP pro- Discussion tein expression level were suitable criteria for evaluating Our hypotheses that BMD, BMC and mRNA or protein dietary Ca requirements of broilers. Based on the best expression levels of ALP, OC, OPG and BMP-2 in bone fitted broken-line or quadratic models of the above cri- might be new sensitive criteria to evaluate dietary Ca re- teria, optimal dietary Ca levels were estimated to be quirements of broilers fed a corn-soybean meal diet, and 1.00%, 0.80%, 0.93%, 0.88%, 0.93%, 0.91% and 0.90% for dietary Ca requirements of broilers from 1 to 21 days of broilers fed a conventional corn-soybean meal diet from age might be different from the current NRC Ca re- 1 to 21 days of age. However, broilers had the highest quirement (1.00%) have been partially supported by the ADG at dietary Ca level of 0.59%. Therefore, in general, results of the current study. The current study indicated Table.5 Effect of dietary calcium (Ca) level on tibia bone parameters of broilers on d 21 Analyzed dietary Ca Tibia ash Tibia ash Ca Tibia ash Tibia ash Ca: P Tibia BMD, Tibia Tibia breaking level, % percentage, % content, % P, % ratio mg/cm BMC, g strength, N bc bc b c 0.59 49.3 35.1 17.8 1.96 136 0.88 98 c c b bc 0.70 48.7 35.3 17.9 1.97 133 0.82 109 ab b ab ab 0.80 50.2 35.6 17.9 1.98 146 0.98 129 a a ab a 0.88 50.9 35.8 18.0 1.99 158 1.00 140 ab b ab ab 0.98 50.6 35.3 17.9 1.97 145 1.02 134 ab bc a ab 1.09 50.5 35.6 17.9 1.99 141 1.10 131 abc bc b ab 1.18 49.4 35.6 17.6 2.03 142 0.95 132 SEM 1.17 0.79 0.34 0.052 8.0 0.105 19.7 P-value Ca level 0.004 0.73 0.40 0.45 0.0003 0.027 0.008 Linear 0.07 –– – 0.11 0.012 0.001 Quadratic 0.003 –– – 0.0006 0.052 0.012 Data represented the means of 6 replicates (n =6) Abbreviations: BMC Bone mineral content, BMD bone mineral density a-c Means within a column with unlike superscript letters were significantly different (P < 0.05) Bai et al. Journal of Animal Science and Biotechnology (2022) 13:11 Page 7 of 12 Table.6 Effect of dietary calcium (Ca) level on middle toe bone parameters of broilers on d 21 Analyzed dietary Ca level, % Toe ash percentage, % Toe ash Ca, % Toe ash P, % Toe ash Ca: P ratio Toe BMD, mg/cm Toe BMC, g c c 0.59 41.6 36.9 17.99 2.04 45 0.13 bc bc 0.70 41.2 37.2 18.14 2.06 51 0.15 abc ab 0.80 41.91 37.6 18.22 2.10 55 0.20 abc a 0.88 42.1 37.6 18.51 2.07 56 0.18 ab ab 0.98 41.9 37.8 18.24 2.06 53 0.18 a c 1.09 42.3 37.9 18.07 2.08 46 0.15 a c 1.18 41.2 38.1 17.99 2.09 46 0.17 SEM 0.215 0.530 0.324 0.053 4.5 0.046 P-value Ca level 0.20 0.008 0.12 0.52 < 0.0001 0.18 Linear – < 0.0001 –– 0.37 – Quadratic – 0.55 –– < 0.0001 – Data represented the means of 6 replicates (n =6) Abbreviations: BMC bone mineral content, BMD bone mineral density a-c Means within a column with unlike superscript letters were significantly different (P < 0.05) that tibia and middle toe BMD, serum and tibia ALP ac- In earlier studies, growth performance was often used tivities, and tibia ALP protein level were new sensitive to assess Ca requirements of broilers [6, 13]. In order to criteria to evaluate dietary Ca requirements of broilers, maximize the growth performance of broilers, using di- and the Ca requirement would be about 0.6% to obtain ets with 0.6% Ca becomes more widespread [2]. As birds the best growth rate and 1.00% to meet all of the Ca me- possess specific Ca appetite [31], they get adapted to low tabolisms and bone development of broilers fed a con- Ca diets by increasing absorption and utilization effi- ventional corn-soybean meal diet from 1 to 21 days of ciency, which decreases excretion of the restricted nutri- age. These findings could better characterize require- ents [12] and elevates plasma 1,25-dihydroxyvitamin D ments and meet the growth, bone development and Ca (1,25(OH) D ) concentration and duodenal calbindin 2 3 metabolic functions of broilers. concentrations [32, 33]. The current study showed that Table.7 Effect of dietary calcium (Ca) level on ALP, OPG, OC and BMP-2 mRNA and protein expression levels in the tibia of broilers on d 21 Analyzed dietary Ca ALP mRNA, OPG mRNA, OC mRNA, BMP-2 mRNA, ALP protein, OPG protein, OC protein, BMP-2 2 2 2 2 3 3 3 3 level, % RQ RQ RQ RQ RQ RQ RQ protein, RQ a ab c 0.59 1.11 0.98 1.69 0.91 0.89 1.14 1.75 1.03 ab a abc 0.70 1.06 1.19 1.24 1.07 1.03 1.03 1.62 0.81 bc bc ab 0.80 0.84 0.97 0.85 0.72 1.17 0.98 1.67 0.79 bc abc a 0.88 0.89 0.85 1.00 0.88 1.19 0.91 1.65 0.73 bc ab abc 0.98 1.00 1.00 1.00 1.00 1.06 0.88 1.88 0.79 c abc bc 1.09 0.87 0.74 0.65 0.79 0.95 1.03 1.58 0.85 c c abc 1.18 0.75 0.79 0.66 0.66 1.03 1.01 1.43 0.89 SEM 0.107 0.113 0.147 0.092 0.069 0.089 0.134 0.081 P-value Ca level 0.37 0.31 0.002 0.02 0.04 0.49 0.37 0.25 Linear –– < 0.0001 0.02 0.75 –– – Quadratic –– 0.11 0.24 0.006 –– – Data represented the means of 6 replicates (n =6) The mRNA levels were calculated as the relative quantity (RQ) of the target gene mRNA to the geometric mean of β-actin and glyceraldehyde-3-phosphate -ΔΔCt dehydrogenase mRNA, RQ = 2 (Ct, threshold cycle) The protein levels were calculated as the relative quantity (RQ) of the target gene protein to the glyceraldehyde-3-phosphate dehydrogenase protein Abbreviations: ALP alkaline phosphatase, BMP-2, morphogenetic protein-2, OC osteocalcin, OPG osteoprotegerin a-c Means within a column with unlike superscript letters were significantly different (P < 0.04) Bai et al. Journal of Animal Science and Biotechnology (2022) 13:11 Page 8 of 12 Table.8 Estimations of dietary calcium (Ca) requirements of broilers from 1 to 21 days of age based on the best fitted broken-line or quadratic models a 2 Dependent variable Regression equation R P- Dietary Ca value requirements, % Serum ALP activity Y = 79.44–79.44X (0.59 ≤ X ≤ 1.00) Y = − 670.1 + 999.9X (1.00 ≤ X ≤ 1.18) 0.49 < 1.00 1 2 0.0001 Tibia ash percentage Y = 44.895 + 6.5766X (0.59 ≤ X ≤ 0.93) Y = −55.9373 - 5.3306X (0.93 ≤ X ≤ 0.29 0.005 0.93 1 2 1.18) Tibia ALP activity Y = 668.4286–473.9648X (0.59 ≤ X ≤ 0.80) Y = 134.7242 + 193.1789X 0.27 0.007 0.80 1 2 (0.80 ≤ X ≤ 1.18) Tibia breaking strength Y = 11.0110 + 145.1498X (0.59 ≤ X ≤ 0.88) Y = 162.1505–27.2524X (0.88 ≤ 0.37 0.0005 0.88 1 2 X ≤ 1.18) Tibia BMD Y = 0.0285 + 0.2592X - 0.1400X 0.24 0.005 0.93 Tibia ALP protein expression Y = −0.6983 + 4.0791X - 2.2713X 0.14 0.021 0.90 level Middle toe BMD Y = −0.0341 + 0.2024X - 0.1153X 0.46 < 0.91 0.0001 Regression equations based on the analyzed Ca concentrations (%) in the diets Abbreviations: ALP alkaline phosphatase, BMD bone mineral density the increase of dietary Ca level from 0.70 to 1.18% had a and 1,25(OH) D play an important role. In addition, 2 3 negative impact on growth rate of broilers from 1 to 21 the Ca content in middle toe ash increased linearly with days. Similar results were reported by Walk et al. [9], in- increasing dietary Ca levels, indicating that middle toe dicating that the increase of dietary Ca level depressed ash Ca is not a useful marker for the assessment of Ca the weight gain and feed intake at a 0.32% dietary NPP requirement of broilers. level. The possible reason for the negative effect of high- Bone characteristics, such as bone ash percentage and Ca diet on broiler’s growth rate might be due to that breaking strength, have been traditional criteria to evalu- relatively high Ca reduced P availability [34], leading to ate bone mineralization in broilers [22]. The NRC Ca the formation of extremely insoluble Ca-phytate com- recommendations for broilers were mainly based on plexes and severe P deficiency. In addition, higher diet- maximizing bone ash. According to the results from the ary Ca could increase intestinal pH, which reduced the current study, the Ca requirements were 0.93% and absorptivity of minerals [35]. Hamdi et al. [13] found 0.88% based on tibia ash percentage and breaking that broilers achieved their greatest weight gain with strength, respectively. And these two criteria increased 0.70% dietary Ca and 0.38% dietary NPP. Additionally, quadratically as dietary Ca increased, which is in line some researchers recommended relatively lower levels of with the results reported by Bar et al. [10]. However, dietary Ca (0.60 to 0.65%) for broilers according to some previous studies showed that the Ca requirement weight gain and feed intake [36, 37], which is similar to (1.3%) based on bone ash contents of 21-d-old broilers the results from the present study. However, Valable was higher than the current NRC Ca requirement et al. [38] and Wilkinson et al. [39] reported that the re- (1.00%) [6, 45], which might be due to the high dietary P duction of dietary Ca did not affect the growth perform- levels (from 0.7 to 0.75%) in these studies. Shafey [46] ance of broilers. Different breeds and growth phases of reported that increasing dietary Ca enhanced the break- broilers might account for the discrepancy of the above ing strength of cockerel tibia. However, Onyango et al. results. [15] did not observe the same phenomenon, and thought The Ca and P contents in serum and bone have been that the high variability in breaking strength contributed considered as good parameters to reflect the nutritional to the lack of statistical significance. Actually, several status of Ca in broilers [40, 41]. However, the current factors can affect breaking strength, such as cross head study showed that dietary Ca level had no effect on speed, handling of bones before testing and measure- serum and tibia Ca, P content and Ca to P ratio, indicat- ment technique [15, 47, 48]. Some researchers deter- ing that these parameters were not suitable to estimate mined BMC and BMD of broilers using DEXA [49, 50]. the Ca requirements. Some researchers reported similar The DEXA technology provided a rapid and noninvasive results [42, 43]. Hurwitz et al. [44] explained that mod- advantage measurement of bone mineralization com- ern broilers had good ability to keep serum Ca and P pared with traditional measurement. Yan et al. [12] contents in narrow range regardless of the varying diet- found that BMC and BMD were good indicators for Ca ary Ca levels mainly because of the oscillations regula- nutrition estimation. Onyango et al. [15] and Valable tion system, in which parathyroid hormone, calcitonin et al. [38] reported that BMC and BMD showed a linear Bai et al. Journal of Animal Science and Biotechnology (2022) 13:11 Page 9 of 12 and quadratic increase with the increase of dietary Ca expression levels in the keel bone of hens. However, in level, which is similar to the results from the present ex- the present study, dietary Ca levels did not affect OPG periment. The optimal Ca level was estimated to be mRNA and OC protein expression levels in the tibia of 0.93% and 0.91% based upon tibia and middle toe BMD, broilers. The disparity may mainly result from the differ- which was close to that (0.93%) estimated by the tibia ent animal species (previous hens vs. present broilers) ash percentage. Onyango et al. [15] showed that there and bone samples. The BMP-2, a potent osteogenic dif- was a high correlation between bone ash and BMC or ferentiation factor, is essential for osteoblast differenti- BMD. The above results indicate that the requirement ation and bone formation [62]. Jia et al. [63] found that for maximizing bone development was greater than that the mRNA and protein levels of BMP-2 increased with 2+ for the best growth performance of broilers, which was increasing Ca concentrations in the primary renal further confirmed by Bar et al. [10]. It might be viable to tubular epithelial cells. A study with piglets demon- reduce the dietary Ca level in order to maximize the strated that the serum BMP-2 concentration exhibited a growth performance of broilers, however, it should not trend from rise to decline with increasing dietary Ca be at the expense of bone health. levels [64]. Similar tendency was observed for the tibia Alkaline phosphatase is a ubiquitous enzyme that cata- BMP-2 mRNA levels as dietary Ca levels increased in lyzes the hydrolysis of phosphate monoesters [51], and can the present study. In addition, the results from the be used as a general indicator of skeletal development [52]. present study showed that as dietary Ca levels increased, The increase of ALP activity is usually associated with inad- the tibia OC and BMP-2 mRNA levels changed linearly, equate supply of Ca or P and poor bone mineralization [53, but not quadratically, indicating that these parameters 54]. Xia et al. [55] found that serum ALP activity was sensi- are not suitable to estimate the requirements of Ca in tive to the dietary Ca, and can be used to evaluate the diet- broilers. We also found that the mRNA levels of OC and ary Ca requirements for laying Longyan shelducks. The BMP-2 in the tibia varied, while their protein levels did current study showed that both serum and tibia ALP activ- not change as dietary Ca levels increased, implying that ities changed quadratically as dietary Ca increased, and the the transcriptional change would often precede the Ca requirements to obtain the minimum ALP activities in translational change [60, 65]. serum and tibia were 1.00% and 0.80%, respectively. Simi- The present study showed that the Ca requirement larly, Hurwitz and Griminger [56]reportedthatserum ALP was 0.59% based on ADG. Similarly, Sebastian et al. [36] activity decreased as dietary Ca increased, reaching a mini- found that the optimum body weight, feed intake, and mum in the range of probable Ca requirement, and could feed efficiency were obtained at 0.60% dietary Ca. We be used as an indicator to evaluate Ca adequacy in growing also found that the Ca requirements of broilers ranged chicks. In the present study, the tibia ALP protein expres- from 0.8 to 1.00% based on tibia and middle toe charac- sion level increased quadratically as dietary Ca increased, teristics, serum or tibia ALP activity, and tibia ALP pro- and the Ca requirement obtained from this indicator was tein expression level. In order to meet all of Ca 0.90%, while the tibia ALP mRNA expression level was not metabolisms and bone development functions, dietary affected by dietary Ca. Mehra et al. [57]explained that Ca requirement would be 1.00% for broilers fed a con- changes in protein levels were not directly related to ventional corn–soybean meal diet from 1 to 21 days of changes in mRNA levels, because of the complexity of tran- age, which is in line with the current NRC Ca require- scription, translation and posttranslational modification. ment. Previous reports suggested that the Ca require- Another consideration might be the time course of the ex- ment should be higher for skeletal development than for periment. The tibia ALP mRNA may have been upregu- optimal growth [10, 66]. Our findings also suggest that lated early in the study in response to dietary treatments, the current NRC (1994) recommendation for Ca (1.00%) and by the end of the study, its expression levels may have would be adequate for the optimum bone development returned to baseline values. but excessive for the best growth rate. Bone metabolism includes bone formation and resorp- tion. Markers of bone formation and resorption act as Conclusions key determinants in the regulation of bone mass [17, The results from the present study indicate that 0.59% 58]. Osteocalcin is the most abundant non-collagenous dietary Ca level was sufficient to obtain the best growth protein of bone matrix and plays an important role in rate of broilers form 1 to 21 days of age. However, con- the bone formation and Ca metabolism [59]. Osteopro- sidering the serum and tibia ALP activities, tibia ash and tegerin is a secreted protein involved in the regulation of breaking strength, tibia and middle toe BMD, and tibia bone resorption [58, 60]. The decrease of OPG mRNA ALP protein expression level, the Ca requirement of expression in the tibia led to the osteoporosis in broilers broilers would be 1.00% to support all of the Ca metabo- [61]. Jiang et al. [19] reported that the increase of dietary lisms and skeletal development, which is the same as the Ca level enhanced OPG mRNA and OC protein current NRC Ca requirement. Bai et al. Journal of Animal Science and Biotechnology (2022) 13:11 Page 10 of 12 Abbreviations 5. Twining PF, Lillie RJ, Robel EJ, Denton CA. Calcium and phosphorus ADFI: Average daily feed intake; ADG: Average daily gain; ALP: Alkaline requirements of broiler chickens. Poult Sci. 1965;44(1):283–96. https://doi. phosphatase; BMC: Bone mineral concentration; BMD: Bone mineral density; org/10.3382/ps.0440283. BMP-2: Bone morphogenetic protein-2; Ca: Calcium; DEXA: Dual-emission X- 6. Yoshida M, Hoshii H. Re-evaluation of requirement of calcium and available ray absorptiometry; 1,25(OH) D : 1,25-dihydroxyvitamin D ; GAPD phosphorus for starting meat-type chicks. Jap Poult Sci. 1982;19(2):101–9. 2 3 3 H: Glyceraldehyde-3-phosphate dehydrogenase; IAS-CAAS: Institute of Animal https://doi.org/10.2141/jpsa.19.101. Science, Chinese Academy of Agricultural Sciences; NPP: Non-phytate 7. Driver JP, Pesti GM, Bakalli RI, Edwards HM Jr. Calcium requirements of the phosphorus; OC: Osteocalcin; OPG: Osteoprotegerin; P: Phosphorus 504 modern broiler chicken as influenced by dietary protein and age. Poult Sci. 2005;84(10):1629–39. https://doi.org/10.1093/ps/84.10.1629. Acknowledgements 8. Havenstein G, Ferket P, Qureshi M. Carcass composition and yield of 1957 The authors gratefully acknowledge all professors and students in the versus 2001 broilers when fed representative 1957 and 2001 broiler diets. mineral nutrition research division. Poult Sci. 2003;82(10):1509–18. https://doi.org/10.1093/ps/82.10.1509. 9. Walk CL, Addo-Chidie EK, Bedford MR, Adeola O. Evaluation of a highly Authors’ contributions soluble calcium source and phytase in the diets of broiler chickens. Poult SB: methodology, data curation, writing-review and editing. YY: investigation, Sci. 2012;91(9):2255–6. https://doi.org/10.3382/ps.2012-02224. writing-original draft preparation. XM: investigation, data curation. XL: valid- 10. Bar A, Shinder S, Yosefi S, Vax E, Plavnik I. Metabolism and requirements for ation, software. RW: writing-review and editing. LZ: project administration. SL: calcium and phosphorus in the fast-growing chicken as affected by age. Br resources. 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Dietary calcium requirements of broilers fed a conventional corn-soybean meal diet from 1 to 21 days of age

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Abstract

Background: The current calcium (Ca) recommendation for broilers is primarily based on studies conducted more than 30 years ago with birds of markedly different productive potentials from those which exist today. And the response indicators in these studies are mainly growth performance and bone ash percentage. Therefore, the present study was carried out to investigate the effect of dietary Ca level on growth performance, serum parameters, bone characteristics and Ca metabolism-related gene expressions, so as to estimate dietary Ca requirements of broilers fed a conventional corn-soybean meal diet from 1 to 21 days of age. Methods: A total of 420 1-day-old Arbor Acres male broilers were randomly assigned to 1 of 7 treatments with 6 replicates (10 birds per cage) and fed the corn-soybean meal diets containing 0.60%, 0.70%, 0.80%, 0.90%, 1.00%, 1.10% or 1.20% Ca for 21 days. Each diet contained the constant non-phytate phosphorus content of about 0.39%. Results: The average daily gain decreased linearly (P < 0.001) as dietary Ca level increased. The serum and tibia alkaline phosphatase (ALP) activities, tibia bone mineral density (BMD), middle toe BMD, tibia ash percentage, tibia breaking strength, and tibia ALP protein expression level were affected (P < 0.05) by dietary Ca level, and showed significant quadratic responses (P < 0.02) to dietary Ca levels. The estimates of dietary Ca requirements were 0.80 to 1.00% based on the best fitted broken-line or quadratic models (P < 0.03) of the above serum and bone parameters, respectively. Conclusions: The results from the present study indicate that the Ca requirements would be about 0.60% to obtain the best growth rate, and 1.00% to meet all of the Ca metabolisms and bone development of broilers fed a conventional corn-soybean meal diet from 1 to 21 days of age. Keywords: Bone characteristic, Broiler, Calcium, Gene expression, Requirement * Correspondence: wlysz@263.net; lulin1225@163.com Shiping Bai and Yunfeng Yang contributed equally to this work. Poultry Mineral Nutrition Laboratory, College of Animal Science and Technology, Yangzhou University, Yangzhou 225000, China Mineral Nutrition Research Division, State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China Full list of author information is available at the end of the article © The Author(s). 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Bai et al. Journal of Animal Science and Biotechnology (2022) 13:11 Page 2 of 12 Background and bone morphogenetic protein-2 (BMP-2) were con- Calcium (Ca) is an essential mineral element, and sidered as good biological markers of bone metabolisms plays an important role in many biological processes, [16–19]. However, the above mentioned indicators have such as enzyme activation, intracellular signaling, never been used to estimate dietary Ca requirements of acid-base balance and bone mineralization [1]. As chickens. We hypothesized that BMD, BMC and mRNA about 99% of Ca is stored in skeleton as hydroxyapa- or protein expression levels of ALP, OC, OPG and tite, Ca is crucial to the bone development of broilers BMP-2 in bone might be new sensitive indices to evalu- [2]. Skeletal abnormalities attract great concern in ate dietary Ca requirements of broilers fed a corn- poultry industryastheycause notonlyproduction soybean meal diet, and dietary Ca requirements of problems but also considerable animal welfare issues. broilers from 1 to 21 days of age might be different from The deficiencies in content or improper ratios of Ca the current NRC Ca requirement (1.00%). and phosphorus (P) were usually considered as main Therefore, the objective of the current study was to in- nutritional reasons leading to greater incidences of leg vestigate the impact of dietary Ca levels on growth per- abnormalities and huge economic loss [3]. formance, serum parameters, bone characteristics and The current Ca recommendations for broilers by Ca metabolism-related gene expressions of broilers, so NRC (1994) [4] were 1.00%, 0.90%, and 0.80% for the as to choose sensitive indices to evaluate dietary Ca re- starter (1 to 21 d), grower (22 to 42 d), and finisher (43 quirements of broilers fed a corn-soybean meal diet to 56 d) phases, respectively. However, these recommen- from 1 to 21 days of age. dations are based on studies conducted in 1960s and 1980s [5, 6], and the response indicators in these studies Materials and methods are mainly growth performance and bone ash percent- All experimental procedures were approved by the Ani- age. The modern broilers differ very much from those mal Management Committee of the Institute of Animal before 1980s in the growth rate, feed conversion effi- Science, Chinese Academy of Agricultural Sciences ciency, carcass quality and bone characteristics [7–9]. (IAS-CAAS, Beijing, China), and performed in accord- The feed efficiency of modern broiler chickens has been ance with the guidelines. Ethical approval on animal sur- improved [7], and it is assumed that the feed intake has vival was given by the animal ethics committee of IAS- decreased, thus the modern broiler chickens have pro- CAAS. The ARRIVE guidelines were followed for report- duced the same adequate bone traits with reduced Ca. ing animal research [20]. Therefore, the Ca requirements of modern fast-growing broilers might be different from those of broiler strains Experimental design, animals and diets reared several decades ago. However, as Ca appears to A total of 420-day-old Arbor Acres male broiler chicks be less threatening to the environment than P [10], and (Huadu Broiler Breeding Corp) with similar body the sources of Ca, namely limestone or oyster shell flour, weights were randomly allotted to 1 of 7 treatments with are cheaper than other mineral sources, the determin- 6 replicate cages of 10 birds per cage in a completely ation of Ca requirements receives little attention [11]. It randomized design, and housed in an electrically heated, has also been reported that modern broilers have a high thermostatically controlled room with fibreglass feeders, capacity to adapt P or Ca deficiency [12]. Nevertheless, waterers and stainless-steel cages coated with plastics for the high level of dietary Ca may reduce the energy value 21 d. They were maintained on a 24-h constant light of the diet and interferes with the availability of other schedule and allowed ad libitum access to experimental minerals [13]. Decreasing the amount of dietary Ca may diets and tap water. The basal corn-soybean meal diet improve performance, but this should not be at the ex- (Table 1) was formulated to meet or exceed the require- pense of increased leg problems [14]. Furthermore, the ments [4, 21] of broilers for all other nutrients except starter phase (1 to 21 d) is a critical period for bone de- for Ca and P. Dietary Ca levels for 7 treatments were velopment of broilers. Thus it is necessary to re-evaluate calculated to be 0.60%, 0.70%, 0.80%, 0.90%, 1.00%, dietary Ca requirements of broilers fed a conventional 1.10% and 1.20% Ca, respectively. The requirement value corn-soybean meal diet from 1 to 21 days of age. of dietary non-phytate P (NPP) recommended by the Chicken bone mineral density (BMD) or content current NRC [4] is 0.45%. In the present study, each diet (BMC) determined by dual-emission X-ray absorpti- contained the constant NPP content of about 0.39% ometry (DEXA) was shown to be highly correlated with based on the results of our previous study [22]. A single bone ash percentage, which could well reflect the Ca nu- large batch of the basal diet without limestone, was tritional status [15]. Furthermore, the enzymes and pro- mixed at first, and then divided into 7 sublots according teins involved in Ca utilization were found sensitive to to the experimental treatment. Each sublot was mixed the dietary Ca levels in animals, especially alkaline phos- with limestone (containing 38.7% Ca by analysis) or fine phatase (ALP), osteocalcin (OC), osteoprotegerin (OPG), sand [23, 24]. The washed fine sand containing no Bai et al. Journal of Animal Science and Biotechnology (2022) 13:11 Page 3 of 12 Table.1 Composition and nutrient levels of the basal diet for collected and stored at − 20 °C for analyses of bone char- broilers from 1 to 21 days of age (as-fed basis) acteristics. The right tibia was snap-frozen in liquid N Item Contents and then stored at − 80 °C for assays of ALP, BMP-2, OC or OPG mRNA and protein expression levels. To re- Ingredients, % duce individual biological variation, samples from two Corn 52.97 chickens in each replicate cage were pooled into one Soybean meal 37.51 sample in equal ratios before analyses, and thus there Soybean oil 5.00 were a total of 6 replicate samples for each treatment. CaHPO 1.72 Limestone 0.24 Serum parameters Salt 0.30 Serum inorganic P was determined by vanadate- DL-Met 0.31 molybdate method [25]. Serum Ca and ALP activities in serum and tibia were measured using commercial assay Micronutrents 0.33 kits (Nanjing Jiancheng Bioengineering Institute, Nan- Sand 1.62 jing, China). Nutrient levels, % ME, MJ/kg 12.69 Bone characteristics Crude protein 21.62 The frozen tibia and middle toe were thawed at room Lysine 1.12 temperature for 2 h, and BMC and BMD were deter- Methionine 0.59 mined by DEXA using the case of small animal model Threonine 0.80 (Lunar_iDXA; GE healthcare, Madison, WI, USA). After the scan, tibia and middle toe were immediately Tryptophane 0.23 rebagged and frozen until further analyses. The tibia Methionine + Cystine 0.90 bone strength was determined using a texture analyser Non-phytate phosphorus 0.39 (TA.XT plus; Stable Micro Systems, London, UK). The Calcium 0.59 tibia was put on a fulcrum point with 50 mm apart. Feed grade Loading point was located in the midpoint of fulcrum Provided per kilogram of diet: VA, 15,000 IU; VD , 4500 IU; VE, 24 IU; VK ,3 3 3 points. The value of breaking force was determined by mg; VB , 3 mg; VB , 9.6 mg; VB , 3 mg;VB , 0.018 mg; Pantothenic acid 1 2 6 12 calcium, 15 mg; Niacin, 39 mg; Folic acid, 1.5 mg; Biotin, 0.15 mg; Choline, 700 the shear test at a speed of 5 mm/min with a 50 kg load- mg; Zn (ZnSO .7H O) 60 mg; Cu (CuSO � 5H O) 8 mg; Mn (MnSO � H O) 110 mg; 4 2 4 2 4 2 ing cell until fracture occurred. The ultimate breaking Fe (FeSO � 7H O) 40 mg; I (KI) 0.35 mg; Se((Na SeO ) 0.35 mg 4 2 2 3 Determined values based on triplicate measurements, and the others were force of the tibia was indirectly obtained, according to calculated values the load v. deformation curve recorded by computer. Following this testing, the tibia and middle toe were detectable Ca and P was used to maintain the same dried at 105 °C for 24 h and defatted with fresh diethyl weight of each treatment diet. The dietary Ca levels by ether for 48 h, and then dried at 105 °C for 12 h to deter- analysis on an as-fed basis were 0.59%, 0.70%, 0.80%, mine bone weight. The dried and defatted tibia and mid- 0.88%, 0.98%, 1.08% and 1.18%, respectively. All diets dle toe were ashed in muffle furnace at 550 °C for 24 h were fed in mash form. At the end of the experiment, to measure bone ash weight and calculate bone ash after fasting for 12-h, broiler weight and feed intake were percentage. recorded for each replicate cage and corrected for mor- tality to calculate average daily gain (ADG), average daily feed intake (ADFI) and feed: gain (FCR) from 1 to 21 Diet and bone ash analyses days of age. Samples of diets and bone ash were ground in a labora- tory mill to pass through a 0.5 mm screen. The crude Sample collections and preparations protein level in the basal diet was analyzed according to At the end of the experiment, chicks in each cage were the Kjeldahl method [26]. The Ca concentrations in diets individually weighed after fasting for 12-h, and 12 chicks and bone ash were determined by inductively coupled (2 chicks per cage) from each treatment were selected plasma spectroscopy (Model IRIS Intrepid II; Thermo according to average body weight of each cage, respect- Jarrell Ash, Waltham, MA, USA). Total P concentrations ively. Blood samples were promptly obtained using wing in diets and bone ash were determined with a spectro- vein puncture, and then centrifuged to harvest serum photometer [27]. Validations of Ca and total P analyses and stored at − 20 °C for analyses of ALP activity and Ca were conducted concurrently using soybean powder or inorganic P contents. Birds were then killed by cer- (GBW10013; National Institute of Standards and Tech- vical dislocation. The left tibia and middle toe were nology, Beijing, China) as a standard reference material. Bai et al. Journal of Animal Science and Biotechnology (2022) 13:11 Page 4 of 12 RNA extraction and quantitative RT-PCR assays gel, and then electrotransferred onto the polyvinylidene The total RNA was isolated from the tibia by using TRI- fluoride membranes (Merck-Millipore, Munich, ZOL reagent (Invitrogen Life Technologies, Carlsbad, Germany). After the transfer, membranes were blocked CA, USA) according to the manufacturer’s instructions. for 1 h at room temperature in a blocking buffer with The concentration of total RNA was estimated by meas- 5% non-fat milk, and then incubated overnight at 4 °C uring its optical density at 260 and 280 nm with a spec- with the following primary antibodies: ALP (A6866; trophotometer (ND-100; NanoDrop Technologies, ABclonal, Wuhan, China), OPG (A135204; ABclonal, Wilmington, DE, USA). A 500 ng of total RNA was re- Wuhan, China), OC (A18241; ABclonal, Wuhan, China), versely transcribed into cDNA using PrimeScript™ RT BMP-2 (A0231; ABclonal, Wuhan, China) and β-tubulin Master Mix Kit (TaKaRa Bio Inc., Otsu, Japan) accord- (HX1829; Huaxingbio, Beijing, China). After washing, ing to the manufacturer’s instructions. The cDNA was the membranes were incubated with the secondary anti- used as templates for real-time quantitative PCR amplifi- body of goat anti-rabbit (HX2028; Huaxingbio, Beijing, cation using SYBR green master mix (Applied Biosys- China) or goat anti-mouse (HX2113; Huaxingbio, tems, Foster City, CA, USA) on the ABI 7500 Real-Time Beijing, China) for 1 h at room temperature. The signals PCR machine following the manufacturer’s guidelines. were recorded with automatic chemiluminescence im- The gene-specific primers for ALP, BMP-2, OPG, OC, β- aging analyzer (5200 Multi; Tanon, Shanghai, China) by actin and glyceraldehyde-3-phosphate dehydrogenase Chemistar ECL Western Blotting Substrate (180–501; (GAPDH) are shown in Table 2. Internal reference Tanon, Shanghai, China). Data were presented as the ra- genes, both β-actin and GAPDH, were constant across tio of ALP, OPG, OC or BMP-2 protein band intensity the dietary treatment groups, and thus their geometric to β-tubulin protein band intensity. mean could be used to normalize the expression of the targeted gene [28]. Relative gene expression was calcu- Statistical analyses 2-ΔΔCt lated using the method [29]. Data from the present study were subjected to one-way ANOVA using the general linear model procedure of Western blotting assays SAS (v. 9.2, SAS Inst. Inc., Cary, NC, USA), and differ- After ground in liquid N , the tibia samples were ho- ences among means were tested by the least significant mogenized in 0.5 mL of ice-cold radio immunoprecipita- difference method. The replicate cage of 9 to 10 chick- tion assay lysis buffer (Beyotime Biotechnology, Haimen, ens for growth performance or two chickens for other China) supplemented with 5 μL of protease inhibitor indicators served as the experimental unit for all statis- (BioTool, Houston, TX, USA). The homogenate was tical analyses. Data from mortality were transformed to centrifuged at 10,000 × g for 10 min at 4 °C, and the arcsine for analysis. Orthogonal comparisons were ap- supernatant was collected for total protein determin- plied for linear and quadratic responses of dependent ation using a BCA Protein Assay Kit (Beyotime Biotech- variables to independent variables. Regression analyses nology, Shanghai, China). The extracted protein (20 μg) of broken-line, quadratic and asymptotic models were was subjected to electrophoresis on a 10% SDS-PAGE performed, and the best fitted models between Table.2 Primer sequences for real-time PCR amplification Gene GenBank ID Primer sequences Length, pb β-actin NM_205518.1 F: 5′-ACCTGAGCGCAAGTACTCTGTCT-3′ 152 R: 5′-CATCGTACTCCTGCTTGCTGAT-3′ GAPDH K01458 F: 5′-CTTTGGCATTGTGGAGGGTC-3′ 128 R: 5′-ACGCTGGGATGATGTTCTGG-3′ ALP NM_205360.1 F: 5′-GGAGAAGGACCCCGAATACTG-3′ 300 R: 5′-TTGACGCCGCAGAGGTAAG-3′ OPG XM_015283019.2 F: 5′-ATCTCAGTCAAGTGGAGCATC-3′ 186 R: 5′-GTTCCAGTCTTCAGCGTAGTA-3′ OC NM_205387.3 F: 5′-TGCTCGCAGTGCTAAAGCCTTCAT-3′ 143 R: 5′-TCAGCTCACACACCTCTCGTT-3′ BMP-2 NM_204358.1 F: 5′-CCAACACCGTGTGCAGCTT-3′ 136 R: 5′-TGGAGTTCAGCTGAGGTGACAGA-3′ Abbreviations: GAPDH glyceraldehyde-3-phosphate dehydrogenase, ALP alkaline phosphatase, OPG osteoprotegerin, OC osteocalcin, BMP-2 bone morphogenetic protein-2 Bai et al. Journal of Animal Science and Biotechnology (2022) 13:11 Page 5 of 12 responsive criteria and dietary Ca level concentrations but tibia BMC increased linearly (P = 0.012). In addition, were used to determine dietary Ca requirements (the tibia breaking strength increased linearly (P = 0.001) and break point from broken-line model or the maximum quadratically (P = 0.012) with increasing dietary Ca response from quadratic model) of broilers [30]. The levels. Tibia ash percentage or breaking strength reached level of statistical significance was set at P ≤ 0.05. a plateau, and tibia BMD reached the highest point at 0.88% Ca. Results Growth performance and mortality Middle toe characteristics Dietary Ca level affected (P< 0.005) body weight on d Dietary Ca level did not influence (P > 0.11) the middle 21 and ADG of broilers from 1 to 21 days of age, but toe ash percentage, P content, Ca to P ratio and BMC, did not affect (P> 0.10) ADFI, FCR and mortality but influenced (P < 0.009) its ash Ca content and BMD (Table 3). The body weight on d 21 and ADG decreased (Table 6). The middle toe ash Ca content increased linearly (P < 0.0001) as dietary Ca level increased. linearly (P < 0.0001), but its BMD increased quadratically Broilers had the highest ADG at dietary Ca level of (P < 0.0001) as dietary Ca level increased. The highest 0.59%. Furthermore, all broilers did not show leg abnor- middle toe BMD was observed visually at 0.88% dietary mality during the whole experimental period. Ca level. Serum parameters and ALP activity in the tibia mRNA levels of ca metabolism-related genes in the tibia Dietary Ca level did not affect (P > 0.05) serum Ca and Dietary Ca level affected (P < 0.03) tibia OC and BMP-2 inorganic P contents, and Ca to inorganic P ratio of mRNA expression levels, but did not affect (P > 0.30) broilers (Table 4), but affected (P < 0.05) ALP activities tibia ALP and OPG mRNA expression levels (Table 7). in serum and tibia. As dietary Ca level increased, the As dietary Ca level increased, both OC and BMP-2 ALP activity in serum increased linearly (P= 0.0003) and mRNA levels decreased linearly (P < 0.03). quadratically (P = 0.002), and it in tibia decreased qua- dratically (P = 0.005). The minimum ALP activity in serum was observed at 0.98% Ca level, and that in tibia Protein expression levels of ca metabolism-related genes at 0.80% Ca level. in the tibia Dietary Ca level affected (P = 0.04) tibia ALP protein ex- Tibia characteristics pression levels, but did not affect (P > 0.24) tibia OPG, Dietary Ca level did not affect (P > 0.39) tibia ash Ca or OC and BMP-2 protein expression levels (Table 7). The P contents and Ca to P ratio, but affected (P < 0.03) the tibia ALP protein expression level increased quadrati- tibia ash percentage, BMD, BMC and breaking strength cally (P = 0.006) as dietary Ca level increased. The high- (Table 5). As dietary Ca level increased, tibia ash per- est tibia ALP protein expression level was observed centage and BMD increased quadratically (P < 0.004), visually at 0.88% dietary Ca level. Table.3 Effect of dietary calcium (Ca) level on the growth performance of broilers during 1 to 21 d of age Analyzed dietary Ca level, % Body weight on d 21, g ADFI, g/d ADG, g/d FCR, g/g Mortality ,% a a 0.59 825 46.32 37.13 1.25 1.67 b b 0.70 793 44.90 35.64 1.26 1.67 bc bc 0.80 781 44.43 35.09 1.27 3.33 b b 0.88 792 44.98 35.58 1.26 0.00 b bc 0.98 789 44.83 35.46 1.27 0.00 bc bc 1.09 768 43.83 34.46 1.27 0.00 c c 1.18 757 43.43 33.94 1.28 0.00 SEM 10.8 1.668 0.096 0.029 0.017 P-value Ca level 0.004 0.11 0.004 0.71 0.33 Linear < 0.0001 – < 0.0001 –– Quadratic 0.72 – 0.72 –– Data represented the means of 6 replicates (n =6) Mortality was based on analysis after anti-sine transform a-c Means within a column with unlike superscript letters were significantly different (P < 0.05) Bai et al. Journal of Animal Science and Biotechnology (2022) 13:11 Page 6 of 12 Table.4 Effect of dietary calcium (Ca) level on serum parameters and tibia ALP activities of broilers on d 21 Analyzed dietary Serum Ca content, Serum inorganic P, Serum Ca: Serum ALP Activity, King Tibia ALP Activity, King Ca level, % mg/100 mL mg/100 mL inorganic P ratio unit/100 mL unit/g prot cd a 0.59 8.63 6.22 1.39 312 384 bc ab 0.70 8.65 7.09 1.23 380 350 cd c 0.80 8.21 6.78 1.22 304 271 bcd bc 0.88 8.50 7.14 1.19 345 305 d ab 0.98 8.95 6.92 1.30 294 340 b ab 1.09 8.63 6.81 1.27 396 348 a ab 1.18 8.76 6.36 1.39 486 355 SEM 0.553 0.534 0.133 51.9 57.1 P-value Ca level 0.43 0.06 0.08 < 0.0001 0.04 Linear –– – 0.0003 0.82 Quadratic –– – 0.002 0.005 Data represented the means of 6 replicates (n =6) Abbreviation: ALP alkaline phosphatase a-d Means within a column with unlike superscript letters were significantly different (P < 0.05) Estimations of dietary ca requirements of broilers the Ca requirement would be about 0.60% to obtain the Results of dietary Ca requirements of broilers as esti- best growth rate and 1.00% to meet all of the Ca metab- mated by the non-linear regression analyses are shown olisms and bone development of broilers fed a conven- in Table 8. The results indicate that serum and tibia tional corn-soybean meal diet from 1 to 21 days of age. ALP activities, tibia ash percentage, tibia breaking strength, tibia and middle toe BMD, and tibia ALP pro- Discussion tein expression level were suitable criteria for evaluating Our hypotheses that BMD, BMC and mRNA or protein dietary Ca requirements of broilers. Based on the best expression levels of ALP, OC, OPG and BMP-2 in bone fitted broken-line or quadratic models of the above cri- might be new sensitive criteria to evaluate dietary Ca re- teria, optimal dietary Ca levels were estimated to be quirements of broilers fed a corn-soybean meal diet, and 1.00%, 0.80%, 0.93%, 0.88%, 0.93%, 0.91% and 0.90% for dietary Ca requirements of broilers from 1 to 21 days of broilers fed a conventional corn-soybean meal diet from age might be different from the current NRC Ca re- 1 to 21 days of age. However, broilers had the highest quirement (1.00%) have been partially supported by the ADG at dietary Ca level of 0.59%. Therefore, in general, results of the current study. The current study indicated Table.5 Effect of dietary calcium (Ca) level on tibia bone parameters of broilers on d 21 Analyzed dietary Ca Tibia ash Tibia ash Ca Tibia ash Tibia ash Ca: P Tibia BMD, Tibia Tibia breaking level, % percentage, % content, % P, % ratio mg/cm BMC, g strength, N bc bc b c 0.59 49.3 35.1 17.8 1.96 136 0.88 98 c c b bc 0.70 48.7 35.3 17.9 1.97 133 0.82 109 ab b ab ab 0.80 50.2 35.6 17.9 1.98 146 0.98 129 a a ab a 0.88 50.9 35.8 18.0 1.99 158 1.00 140 ab b ab ab 0.98 50.6 35.3 17.9 1.97 145 1.02 134 ab bc a ab 1.09 50.5 35.6 17.9 1.99 141 1.10 131 abc bc b ab 1.18 49.4 35.6 17.6 2.03 142 0.95 132 SEM 1.17 0.79 0.34 0.052 8.0 0.105 19.7 P-value Ca level 0.004 0.73 0.40 0.45 0.0003 0.027 0.008 Linear 0.07 –– – 0.11 0.012 0.001 Quadratic 0.003 –– – 0.0006 0.052 0.012 Data represented the means of 6 replicates (n =6) Abbreviations: BMC Bone mineral content, BMD bone mineral density a-c Means within a column with unlike superscript letters were significantly different (P < 0.05) Bai et al. Journal of Animal Science and Biotechnology (2022) 13:11 Page 7 of 12 Table.6 Effect of dietary calcium (Ca) level on middle toe bone parameters of broilers on d 21 Analyzed dietary Ca level, % Toe ash percentage, % Toe ash Ca, % Toe ash P, % Toe ash Ca: P ratio Toe BMD, mg/cm Toe BMC, g c c 0.59 41.6 36.9 17.99 2.04 45 0.13 bc bc 0.70 41.2 37.2 18.14 2.06 51 0.15 abc ab 0.80 41.91 37.6 18.22 2.10 55 0.20 abc a 0.88 42.1 37.6 18.51 2.07 56 0.18 ab ab 0.98 41.9 37.8 18.24 2.06 53 0.18 a c 1.09 42.3 37.9 18.07 2.08 46 0.15 a c 1.18 41.2 38.1 17.99 2.09 46 0.17 SEM 0.215 0.530 0.324 0.053 4.5 0.046 P-value Ca level 0.20 0.008 0.12 0.52 < 0.0001 0.18 Linear – < 0.0001 –– 0.37 – Quadratic – 0.55 –– < 0.0001 – Data represented the means of 6 replicates (n =6) Abbreviations: BMC bone mineral content, BMD bone mineral density a-c Means within a column with unlike superscript letters were significantly different (P < 0.05) that tibia and middle toe BMD, serum and tibia ALP ac- In earlier studies, growth performance was often used tivities, and tibia ALP protein level were new sensitive to assess Ca requirements of broilers [6, 13]. In order to criteria to evaluate dietary Ca requirements of broilers, maximize the growth performance of broilers, using di- and the Ca requirement would be about 0.6% to obtain ets with 0.6% Ca becomes more widespread [2]. As birds the best growth rate and 1.00% to meet all of the Ca me- possess specific Ca appetite [31], they get adapted to low tabolisms and bone development of broilers fed a con- Ca diets by increasing absorption and utilization effi- ventional corn-soybean meal diet from 1 to 21 days of ciency, which decreases excretion of the restricted nutri- age. These findings could better characterize require- ents [12] and elevates plasma 1,25-dihydroxyvitamin D ments and meet the growth, bone development and Ca (1,25(OH) D ) concentration and duodenal calbindin 2 3 metabolic functions of broilers. concentrations [32, 33]. The current study showed that Table.7 Effect of dietary calcium (Ca) level on ALP, OPG, OC and BMP-2 mRNA and protein expression levels in the tibia of broilers on d 21 Analyzed dietary Ca ALP mRNA, OPG mRNA, OC mRNA, BMP-2 mRNA, ALP protein, OPG protein, OC protein, BMP-2 2 2 2 2 3 3 3 3 level, % RQ RQ RQ RQ RQ RQ RQ protein, RQ a ab c 0.59 1.11 0.98 1.69 0.91 0.89 1.14 1.75 1.03 ab a abc 0.70 1.06 1.19 1.24 1.07 1.03 1.03 1.62 0.81 bc bc ab 0.80 0.84 0.97 0.85 0.72 1.17 0.98 1.67 0.79 bc abc a 0.88 0.89 0.85 1.00 0.88 1.19 0.91 1.65 0.73 bc ab abc 0.98 1.00 1.00 1.00 1.00 1.06 0.88 1.88 0.79 c abc bc 1.09 0.87 0.74 0.65 0.79 0.95 1.03 1.58 0.85 c c abc 1.18 0.75 0.79 0.66 0.66 1.03 1.01 1.43 0.89 SEM 0.107 0.113 0.147 0.092 0.069 0.089 0.134 0.081 P-value Ca level 0.37 0.31 0.002 0.02 0.04 0.49 0.37 0.25 Linear –– < 0.0001 0.02 0.75 –– – Quadratic –– 0.11 0.24 0.006 –– – Data represented the means of 6 replicates (n =6) The mRNA levels were calculated as the relative quantity (RQ) of the target gene mRNA to the geometric mean of β-actin and glyceraldehyde-3-phosphate -ΔΔCt dehydrogenase mRNA, RQ = 2 (Ct, threshold cycle) The protein levels were calculated as the relative quantity (RQ) of the target gene protein to the glyceraldehyde-3-phosphate dehydrogenase protein Abbreviations: ALP alkaline phosphatase, BMP-2, morphogenetic protein-2, OC osteocalcin, OPG osteoprotegerin a-c Means within a column with unlike superscript letters were significantly different (P < 0.04) Bai et al. Journal of Animal Science and Biotechnology (2022) 13:11 Page 8 of 12 Table.8 Estimations of dietary calcium (Ca) requirements of broilers from 1 to 21 days of age based on the best fitted broken-line or quadratic models a 2 Dependent variable Regression equation R P- Dietary Ca value requirements, % Serum ALP activity Y = 79.44–79.44X (0.59 ≤ X ≤ 1.00) Y = − 670.1 + 999.9X (1.00 ≤ X ≤ 1.18) 0.49 < 1.00 1 2 0.0001 Tibia ash percentage Y = 44.895 + 6.5766X (0.59 ≤ X ≤ 0.93) Y = −55.9373 - 5.3306X (0.93 ≤ X ≤ 0.29 0.005 0.93 1 2 1.18) Tibia ALP activity Y = 668.4286–473.9648X (0.59 ≤ X ≤ 0.80) Y = 134.7242 + 193.1789X 0.27 0.007 0.80 1 2 (0.80 ≤ X ≤ 1.18) Tibia breaking strength Y = 11.0110 + 145.1498X (0.59 ≤ X ≤ 0.88) Y = 162.1505–27.2524X (0.88 ≤ 0.37 0.0005 0.88 1 2 X ≤ 1.18) Tibia BMD Y = 0.0285 + 0.2592X - 0.1400X 0.24 0.005 0.93 Tibia ALP protein expression Y = −0.6983 + 4.0791X - 2.2713X 0.14 0.021 0.90 level Middle toe BMD Y = −0.0341 + 0.2024X - 0.1153X 0.46 < 0.91 0.0001 Regression equations based on the analyzed Ca concentrations (%) in the diets Abbreviations: ALP alkaline phosphatase, BMD bone mineral density the increase of dietary Ca level from 0.70 to 1.18% had a and 1,25(OH) D play an important role. In addition, 2 3 negative impact on growth rate of broilers from 1 to 21 the Ca content in middle toe ash increased linearly with days. Similar results were reported by Walk et al. [9], in- increasing dietary Ca levels, indicating that middle toe dicating that the increase of dietary Ca level depressed ash Ca is not a useful marker for the assessment of Ca the weight gain and feed intake at a 0.32% dietary NPP requirement of broilers. level. The possible reason for the negative effect of high- Bone characteristics, such as bone ash percentage and Ca diet on broiler’s growth rate might be due to that breaking strength, have been traditional criteria to evalu- relatively high Ca reduced P availability [34], leading to ate bone mineralization in broilers [22]. The NRC Ca the formation of extremely insoluble Ca-phytate com- recommendations for broilers were mainly based on plexes and severe P deficiency. In addition, higher diet- maximizing bone ash. According to the results from the ary Ca could increase intestinal pH, which reduced the current study, the Ca requirements were 0.93% and absorptivity of minerals [35]. Hamdi et al. [13] found 0.88% based on tibia ash percentage and breaking that broilers achieved their greatest weight gain with strength, respectively. And these two criteria increased 0.70% dietary Ca and 0.38% dietary NPP. Additionally, quadratically as dietary Ca increased, which is in line some researchers recommended relatively lower levels of with the results reported by Bar et al. [10]. However, dietary Ca (0.60 to 0.65%) for broilers according to some previous studies showed that the Ca requirement weight gain and feed intake [36, 37], which is similar to (1.3%) based on bone ash contents of 21-d-old broilers the results from the present study. However, Valable was higher than the current NRC Ca requirement et al. [38] and Wilkinson et al. [39] reported that the re- (1.00%) [6, 45], which might be due to the high dietary P duction of dietary Ca did not affect the growth perform- levels (from 0.7 to 0.75%) in these studies. Shafey [46] ance of broilers. Different breeds and growth phases of reported that increasing dietary Ca enhanced the break- broilers might account for the discrepancy of the above ing strength of cockerel tibia. However, Onyango et al. results. [15] did not observe the same phenomenon, and thought The Ca and P contents in serum and bone have been that the high variability in breaking strength contributed considered as good parameters to reflect the nutritional to the lack of statistical significance. Actually, several status of Ca in broilers [40, 41]. However, the current factors can affect breaking strength, such as cross head study showed that dietary Ca level had no effect on speed, handling of bones before testing and measure- serum and tibia Ca, P content and Ca to P ratio, indicat- ment technique [15, 47, 48]. Some researchers deter- ing that these parameters were not suitable to estimate mined BMC and BMD of broilers using DEXA [49, 50]. the Ca requirements. Some researchers reported similar The DEXA technology provided a rapid and noninvasive results [42, 43]. Hurwitz et al. [44] explained that mod- advantage measurement of bone mineralization com- ern broilers had good ability to keep serum Ca and P pared with traditional measurement. Yan et al. [12] contents in narrow range regardless of the varying diet- found that BMC and BMD were good indicators for Ca ary Ca levels mainly because of the oscillations regula- nutrition estimation. Onyango et al. [15] and Valable tion system, in which parathyroid hormone, calcitonin et al. [38] reported that BMC and BMD showed a linear Bai et al. Journal of Animal Science and Biotechnology (2022) 13:11 Page 9 of 12 and quadratic increase with the increase of dietary Ca expression levels in the keel bone of hens. However, in level, which is similar to the results from the present ex- the present study, dietary Ca levels did not affect OPG periment. The optimal Ca level was estimated to be mRNA and OC protein expression levels in the tibia of 0.93% and 0.91% based upon tibia and middle toe BMD, broilers. The disparity may mainly result from the differ- which was close to that (0.93%) estimated by the tibia ent animal species (previous hens vs. present broilers) ash percentage. Onyango et al. [15] showed that there and bone samples. The BMP-2, a potent osteogenic dif- was a high correlation between bone ash and BMC or ferentiation factor, is essential for osteoblast differenti- BMD. The above results indicate that the requirement ation and bone formation [62]. Jia et al. [63] found that for maximizing bone development was greater than that the mRNA and protein levels of BMP-2 increased with 2+ for the best growth performance of broilers, which was increasing Ca concentrations in the primary renal further confirmed by Bar et al. [10]. It might be viable to tubular epithelial cells. A study with piglets demon- reduce the dietary Ca level in order to maximize the strated that the serum BMP-2 concentration exhibited a growth performance of broilers, however, it should not trend from rise to decline with increasing dietary Ca be at the expense of bone health. levels [64]. Similar tendency was observed for the tibia Alkaline phosphatase is a ubiquitous enzyme that cata- BMP-2 mRNA levels as dietary Ca levels increased in lyzes the hydrolysis of phosphate monoesters [51], and can the present study. In addition, the results from the be used as a general indicator of skeletal development [52]. present study showed that as dietary Ca levels increased, The increase of ALP activity is usually associated with inad- the tibia OC and BMP-2 mRNA levels changed linearly, equate supply of Ca or P and poor bone mineralization [53, but not quadratically, indicating that these parameters 54]. Xia et al. [55] found that serum ALP activity was sensi- are not suitable to estimate the requirements of Ca in tive to the dietary Ca, and can be used to evaluate the diet- broilers. We also found that the mRNA levels of OC and ary Ca requirements for laying Longyan shelducks. The BMP-2 in the tibia varied, while their protein levels did current study showed that both serum and tibia ALP activ- not change as dietary Ca levels increased, implying that ities changed quadratically as dietary Ca increased, and the the transcriptional change would often precede the Ca requirements to obtain the minimum ALP activities in translational change [60, 65]. serum and tibia were 1.00% and 0.80%, respectively. Simi- The present study showed that the Ca requirement larly, Hurwitz and Griminger [56]reportedthatserum ALP was 0.59% based on ADG. Similarly, Sebastian et al. [36] activity decreased as dietary Ca increased, reaching a mini- found that the optimum body weight, feed intake, and mum in the range of probable Ca requirement, and could feed efficiency were obtained at 0.60% dietary Ca. We be used as an indicator to evaluate Ca adequacy in growing also found that the Ca requirements of broilers ranged chicks. In the present study, the tibia ALP protein expres- from 0.8 to 1.00% based on tibia and middle toe charac- sion level increased quadratically as dietary Ca increased, teristics, serum or tibia ALP activity, and tibia ALP pro- and the Ca requirement obtained from this indicator was tein expression level. In order to meet all of Ca 0.90%, while the tibia ALP mRNA expression level was not metabolisms and bone development functions, dietary affected by dietary Ca. Mehra et al. [57]explained that Ca requirement would be 1.00% for broilers fed a con- changes in protein levels were not directly related to ventional corn–soybean meal diet from 1 to 21 days of changes in mRNA levels, because of the complexity of tran- age, which is in line with the current NRC Ca require- scription, translation and posttranslational modification. ment. Previous reports suggested that the Ca require- Another consideration might be the time course of the ex- ment should be higher for skeletal development than for periment. The tibia ALP mRNA may have been upregu- optimal growth [10, 66]. Our findings also suggest that lated early in the study in response to dietary treatments, the current NRC (1994) recommendation for Ca (1.00%) and by the end of the study, its expression levels may have would be adequate for the optimum bone development returned to baseline values. but excessive for the best growth rate. Bone metabolism includes bone formation and resorp- tion. Markers of bone formation and resorption act as Conclusions key determinants in the regulation of bone mass [17, The results from the present study indicate that 0.59% 58]. Osteocalcin is the most abundant non-collagenous dietary Ca level was sufficient to obtain the best growth protein of bone matrix and plays an important role in rate of broilers form 1 to 21 days of age. However, con- the bone formation and Ca metabolism [59]. Osteopro- sidering the serum and tibia ALP activities, tibia ash and tegerin is a secreted protein involved in the regulation of breaking strength, tibia and middle toe BMD, and tibia bone resorption [58, 60]. The decrease of OPG mRNA ALP protein expression level, the Ca requirement of expression in the tibia led to the osteoporosis in broilers broilers would be 1.00% to support all of the Ca metabo- [61]. Jiang et al. [19] reported that the increase of dietary lisms and skeletal development, which is the same as the Ca level enhanced OPG mRNA and OC protein current NRC Ca requirement. Bai et al. Journal of Animal Science and Biotechnology (2022) 13:11 Page 10 of 12 Abbreviations 5. Twining PF, Lillie RJ, Robel EJ, Denton CA. Calcium and phosphorus ADFI: Average daily feed intake; ADG: Average daily gain; ALP: Alkaline requirements of broiler chickens. Poult Sci. 1965;44(1):283–96. https://doi. phosphatase; BMC: Bone mineral concentration; BMD: Bone mineral density; org/10.3382/ps.0440283. BMP-2: Bone morphogenetic protein-2; Ca: Calcium; DEXA: Dual-emission X- 6. Yoshida M, Hoshii H. Re-evaluation of requirement of calcium and available ray absorptiometry; 1,25(OH) D : 1,25-dihydroxyvitamin D ; GAPD phosphorus for starting meat-type chicks. Jap Poult Sci. 1982;19(2):101–9. 2 3 3 H: Glyceraldehyde-3-phosphate dehydrogenase; IAS-CAAS: Institute of Animal https://doi.org/10.2141/jpsa.19.101. Science, Chinese Academy of Agricultural Sciences; NPP: Non-phytate 7. Driver JP, Pesti GM, Bakalli RI, Edwards HM Jr. Calcium requirements of the phosphorus; OC: Osteocalcin; OPG: Osteoprotegerin; P: Phosphorus 504 modern broiler chicken as influenced by dietary protein and age. Poult Sci. 2005;84(10):1629–39. https://doi.org/10.1093/ps/84.10.1629. Acknowledgements 8. Havenstein G, Ferket P, Qureshi M. Carcass composition and yield of 1957 The authors gratefully acknowledge all professors and students in the versus 2001 broilers when fed representative 1957 and 2001 broiler diets. mineral nutrition research division. Poult Sci. 2003;82(10):1509–18. https://doi.org/10.1093/ps/82.10.1509. 9. Walk CL, Addo-Chidie EK, Bedford MR, Adeola O. Evaluation of a highly Authors’ contributions soluble calcium source and phytase in the diets of broiler chickens. Poult SB: methodology, data curation, writing-review and editing. YY: investigation, Sci. 2012;91(9):2255–6. https://doi.org/10.3382/ps.2012-02224. writing-original draft preparation. XM: investigation, data curation. XL: valid- 10. Bar A, Shinder S, Yosefi S, Vax E, Plavnik I. Metabolism and requirements for ation, software. RW: writing-review and editing. LZ: project administration. SL: calcium and phosphorus in the fast-growing chicken as affected by age. Br resources. 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Journal

Journal of Animal Science and Biotechnology – Springer Journals

Published: Feb 3, 2022

Keywords: Bone characteristic; Broiler; Calcium; Gene expression; Requirement

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