High calcium to phosphorus ratio impairs growth and bone mineralization in Pekin ducklings

High calcium to phosphorus ratio impairs growth and bone mineralization in Pekin ducklings Abstract Two experiments were conducted to investigate the effect of high dietary calcium (Ca) level on growth performance, Ca and phosphorus (P) metabolism, and nutrient utilization in ducklings subjected to normal and low P levels in diets. A completely randomized design was used with a factorial arrangement of 2 total dietary P levels [normal-P (0.60%) and low-P (0.45%) groups] × 4 dietary Ca levels [low-Ca (0.55%), normal-Ca (0.75%), medium-Ca (0.95%) and high-Ca (1.15%) groups)]. Compared to normal-P group, low-P group had lower (P < 0.05) final body weight (BW), average daily gain (ADG), and average daily feed intake (ADFI) and reduced (P < 0.05) serum Ca and P levels, bone Ca, P, and ash content, and bone mineral density in ducklings during the starter period. Under the low-P group, birds from high-Ca group had lower (P < 0.05) final BW, ADG, ADFI, bone ash content, bone mineral density, and the utilization of energy, Ca, and P than those from low-Ca, normal-Ca, and medium-Ca groups. Our results indicate that high-Ca diet induced greater growth suppression and bone mineralization loss in ducklings fed a low-P diet. The aggravated negative effect of high dietary Ca level with a low P level might be related to the elevated serum alkaline phosphatase activity and the reduced utilization of energy, Ca, and P. INTRODUCTION Calcium (Ca) is an essential mineral nutrient for optimal growth and bone development in poultry (Atteh and Leeson, 1984). The growth of modern meat-breed ducks has been accelerated during recent decades (Farrell, 1990). The NRC (1994) recommendation of Ca (0.60∼0.65%) for ducks, based on a 1967 study (Dean et al., 1967), may not be applicable for modern ducks. In practical production, a tendency to over-supplement (0.85∼1.10%) Ca as an insurance factor to prevent skeletal problems and leg abnormalities has been adopted for fast-growing strains of ducks (Applegate and Angel, 2014; Roberson et al., 2004). Previous studies in broilers showed that daily weight gain and bone ash content were increased as dietary Ca content increased from 0.40 to 0.90% (Driver et al., 2005). However, when dietary Ca levels are above the Ca requirement, it might impair growth performance and decrease bone mineralization in broilers (Rao et al., 2006; Hamdi et al., 2015). There must be recognition of the possible adverse effects of high Ca intake on the metabolism of other mineral nutrients (Schiller et al., 1989; Wood and Zheng, 1997; Minihane and Fairweather-Tait, 1998). High Ca intake induced a significant reduction in the efficiency of phosphorus (P) absorption in poultry (Simpson and Wise, 1990). Excess Ca can interact with inorganic P to form the insoluble complexes (Schiller et al., 1989), which reduced the utilization of Ca and P (Plumstead et al., 2008). So far, the negative effects of high dietary Ca on duck performance have not been well demonstrated. It is assumed that the deleterious effect of high Ca intake would be even more evident at limited amounts of available P or P deficiency in the diets. Therefore, the objective of this study was to investigate the effect of high dietary Ca level on the growth performance, characteristics of serum and tibia bone, and nutrient utilization in ducklings subjected to the normal-P and low-P level diets. MATERIALS AND METHODS Animals and Diets All experimental procedures were approved by the Institutional Animal Care and Use Committee of South China Agricultural University. The study included feeding (Exp. 1) and metabolic experiments with ducks (Exp. 2). In Exp. 1, 960 day-old male Cherry Valley ducklings were weighed individually and allotted to 8 dietary treatments. Each treatment was 4 replicated pen with 30 ducklings per pen. Feed and water were provided ad libitum from hatch to d 21 during the experimental period. The experimental design was completely randomized with a 2 × 4 factorial arrangement of treatments. These variables included 2 total dietary P levels at 0.60% (normal-P group) and 0.45% (low-P group) and 4 dietary Ca levels at 0.55% (low-Ca group), 0.75% (normal-Ca group), 0.95% (medium-Ca group), 1.15% (high-Ca group). The basal diets were formulated to meet or exceed nutrition requirements recommended by NRC (1994) for ducklings at the starter period except Ca and P. Composition and nutrient levels of the experimental diets were presented in Table 1. At 21 d of age, after 12 h feed withdrawal, birds were weighed and feed consumption and mortality were recorded by each replicate pen. The average daily gain (ADG), average daily feed intake (ADFI) and feed/gain ratio (F/G) were calculated. Based on the average body weight (BW) of birds in each replicate pen, 2 birds in each pen were taken for blood sampling, and then were euthanized by CO2 inhalation, and the tibias were removed and used for measuring the indices related to bone characteristics. In Exp. 2, the Sibbald method described by McNab and Blair (1988) was used to evaluate the utilization of energy, CP, Ca, and P of 8 diets used in Exp. 1 for ducks. Briefly, 6 male Cherry Valley ducks, age 10 wk, of similar weight (3.7 to 3.8 kg/bird) were assigned to each of 8 test diets. As described previously (Adeola et al., 1997; King et al., 1997), during the adaptation period of the first 48 h, all the birds were intubated with dextrose solution (30 g/100 mL of water) at 8 and 32 h after feed was removed. Thirty grams (30 g/100 mL of water) of each diet were tube-fed to each bird at 48 h after feed was withdrawn. In order to reduce excessive weight loss and the variability in endogenous nutrient loss (ENL), another 6 ducks that served as controls for estimation of endogenous losses of Ca, P, CP and energy were tube-fed 30 g dextrose (30 g/100 mL of water) at 48 h after feed was withdrawn (Adeola et al., 1997). Total excreta samples were collected during the next 48 h from each bird using Playtex bottles with attached Whirl-pak bags (McNab and Blair, 1988). As described by Adeola et al. (1997), the standardized values of nutrient utilization (NU) in the experimental diets were calculated using the following formula: NU = (NI–NO + ENL)/NI × 100%, where NI = nutrient intake of the diet; NO = nutrient output of the excreta; ENL = endogenous nutrient loss. Table 1. Composition and nutrient levels of the experimental diets (as-fed basis). Total P level, %  0.60  0.45  Ca level, %  0.55  0.75  0.95  1.15  0.55  0.75  0.95  1.15  Corn  63.60  63.40  63.08  63.00  63.65  63.60  63.43  63.01  Soybean meal  23.5  22.5  21.5  22.0  24.0  24.0  23.0  23.5  Corn gluten meal  5.60  6.25  7.00  7.80  4.70  5.40  6.50  7.00  Cottonseed meal  2.00  2.00  2.00  1.26  2.30  1.60  1.10  1.00  Rapeseed meal  2.00  2.00  2.00  1.00  2.50  2.00  2.00  1.00  L-lysine·HCl  0.40  0.42  0.45  0.45  0.39  0.39  0.42  0.42  DL-Methionine  0.15  0.15  0.15  0.14  0.16  0.16  0.15  0.15  Dicalcium phosphate  1.65  1.65  1.65  1.65  0.70  0.75  0.75  0.75  Sodium chloride  0.30  0.30  0.30  0.30  0.30  0.30  0.30  0.30  Limestone  0.30  0.83  1.37  1.90  0.80  1.30  1.85  2.37  Vitamin and mineral premix1  0.50  0.50  0.50  0.50  0.50  0.50  0.50  0.50  Total  100  100  100  100  100  100  100  100  Nutrient composition                  Calculated value, %                  ME, kcal/kg  2895  2895  2895  2895  2895  2895  2895  2895  Crude protein  20.5  20.5  20.5  20.5  20.5  20.5  20.5  20.5  Lysine  1.17  1.17  1.17  1.17  1.17  1.17  1.17  1.17  Methionine  0.48  0.48  0.48  0.48  0.48  0.48  0.48  0.48  Methionine + cysteine  0.81  0.81  0.81  0.81  0.81  0.81  0.81  0.81  Calcium  0.55  0.75  0.95  1.15  0.55  0.75  0.95  1.15  Total phosphorus  0.60  0.60  0.60  0.60  0.45  0.45  0.45  0.45  Analyzed value2, %                  Calcium  0.60  0.78  0.95  1.15  0.63  0.78  0.93  1.20  Total phosphorus  0.57  0.61  0.61  0.60  0.45  0.50  0.49  0.47  Total P level, %  0.60  0.45  Ca level, %  0.55  0.75  0.95  1.15  0.55  0.75  0.95  1.15  Corn  63.60  63.40  63.08  63.00  63.65  63.60  63.43  63.01  Soybean meal  23.5  22.5  21.5  22.0  24.0  24.0  23.0  23.5  Corn gluten meal  5.60  6.25  7.00  7.80  4.70  5.40  6.50  7.00  Cottonseed meal  2.00  2.00  2.00  1.26  2.30  1.60  1.10  1.00  Rapeseed meal  2.00  2.00  2.00  1.00  2.50  2.00  2.00  1.00  L-lysine·HCl  0.40  0.42  0.45  0.45  0.39  0.39  0.42  0.42  DL-Methionine  0.15  0.15  0.15  0.14  0.16  0.16  0.15  0.15  Dicalcium phosphate  1.65  1.65  1.65  1.65  0.70  0.75  0.75  0.75  Sodium chloride  0.30  0.30  0.30  0.30  0.30  0.30  0.30  0.30  Limestone  0.30  0.83  1.37  1.90  0.80  1.30  1.85  2.37  Vitamin and mineral premix1  0.50  0.50  0.50  0.50  0.50  0.50  0.50  0.50  Total  100  100  100  100  100  100  100  100  Nutrient composition                  Calculated value, %                  ME, kcal/kg  2895  2895  2895  2895  2895  2895  2895  2895  Crude protein  20.5  20.5  20.5  20.5  20.5  20.5  20.5  20.5  Lysine  1.17  1.17  1.17  1.17  1.17  1.17  1.17  1.17  Methionine  0.48  0.48  0.48  0.48  0.48  0.48  0.48  0.48  Methionine + cysteine  0.81  0.81  0.81  0.81  0.81  0.81  0.81  0.81  Calcium  0.55  0.75  0.95  1.15  0.55  0.75  0.95  1.15  Total phosphorus  0.60  0.60  0.60  0.60  0.45  0.45  0.45  0.45  Analyzed value2, %                  Calcium  0.60  0.78  0.95  1.15  0.63  0.78  0.93  1.20  Total phosphorus  0.57  0.61  0.61  0.60  0.45  0.50  0.49  0.47  1Provided per kilogram of diet: vitamin A, 4,000 IU; vitamin D3, 2,000 IU; vitamin E, 24 IU; thiamine, 2.0 mg; riboflavin, 12 mg; pyridoxine, 4.0 mg; vitamin B12, 0.02 mg; calcium pantothenate, 10 mg; folate, 0.15 mg; niacin, 50 mg; biotin, 0.15 mg; choline (Choline chloride), 1,000 mg; Cu (CuSO4·5H2O), 8 mg; Fe (FeSO4·7H2O), 80 mg; Zn (ZnSO4·7H2O), 90 mg; Mn (MnSO4·H2O), 70 mg; Se (NaSeO3), 0.3 mg; I (KI), 0.4 mg. 2Analysed values based on triplicate determinations. View Large Sample Collections and Preparations Blood samples were obtained via a bronchial vein (3.5 mL/bird), immediately placed on ice, and then centrifuged at 2,000 × g for 15 min in a refrigerated centrifuge to prepare serum. The serum samples were stored at −20°C for the analysis of Ca and P concentrations and other biochemical parameters. Tibia bones were removed and then were dried at 105°C for 24 h, and then defatted 48 h in ethyl alcohol followed by a 48 h extraction in ethyl ether, and then dried for 12 h at 110°C. Tibia bone ash percentage were determined by ashing overnight at 550°C with a muffle furnace. Diets, bone ash, and freeze-dried excreta samples were analyzed for crude protein (CP) (method 988.05; Association of Official Analytical Chemists (AOAC), 1990), and Ca and total P (method 985.01; AOAC, 1990). The bone mineral density (BMD) were measured by DEXA (DCS-600; Aloka, Tokyo, Japan) as described by Liu et al. (2017). Gross energy was determined using an adiabatic calorimetric bomb (IKA C200; Heitersheim, Germany). The Ca and P concentrations and alkaline phosphatase (ALP) activity in serum was determined by HITACHI 7180 automatic biochemical analyzer (Hitachi Ltd., Tokyo, Japan) with commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Serum calcitonin (CT) was measured by radioimmunoassay using commercial kits (Beijing North Institute of Biological Technology, Beijing, China). Statistical Analyses All data were analyzed by 2-way analysis of variance (ANOVA) using the general linear model procedure of SAS 9.2 (SAS Institute, 2010). The model included the main effects of dietary P, dietary Ca, and their interactions. Each replicate cage (Exp.1) or duck (Exp.2) served as an experimental unit. Data from mortality of ducklings were transformed to arcsin before statistical analysis. Differences among means were tested by the Least Square Difference method, and statistical significance was set at P < 0.05. RESULTS Growth Performance Dietary P, dietary Ca, and their interactions affect the final BW (P < 0.0001), ADG (P < 0.0001) and ADFI (P < 0.05) of ducklings from hatch to d 21 (Table 2). The F/G and mortality was affected (P < 0.05) by dietary P, but not by dietary Ca (P > 0.05) and their interactions (P > 0.05; Table 1). The high-Ca diet decreased (P < 0.05) the final BW, ADG, and ADFI of ducklings from hatch to d 21 compared to the other 3 Ca diets. Compared to normal-P group, low-P group decreased the final BW, ADG, and ADFI, and increased F/G and mortality of ducklings from hatch to d 21. Under the normal-P, no differences (P > 0.05) were observed in the final BW, ADG, and ADFI of ducklings as dietary Ca increased from 0.55 to 0.95%; under the low-P group, however, final BW, ADG, and ADFI were decreased (P < 0.05) as dietary Ca level increased from 0.55 to 1.15%. Table 2. Effect of dietary Ca level on growth performance and mortality of ducklings subjected to the normal and low P diets from hatch to d 21 in Exp. 1. TP  Ca    Final BW,  ADG,  ADFI,  F/G,  Mortality,  level, %  level, %  Reps  g  g/d/bird  g/d/bird  g/g  %  0.60  0.55  4  1248a,b  56.9b,c  97.3a  1.71  10.0    0.75  4  1283a  58.5a,b  97.5a  1.67  0.0    0.95  4  1250a,b  56.7b,c  98.4a  1.73  2.5    1.15  4  1190b,c  54.2c  92.8a,b  1.72  0.8  0.45  0.55  4  1072d  48.5d  87.8b,c  1.82  14.2    0.75  4  1055d  47.7d  85.4c,d  1.81  4.2    0.95  4  1014d  45.7d  78.8d  1.72  10.0    1.15  4  750e  33.2e  65.4e  2.18  22.5  Pooled SEM    23  1.1  2.9  0.10  4.5  TP, %  0.60  16  1248  56.6  96.5  1.71b  3.3b  0.45  16  973  43.7  79.3  1.88a  12.7a  Pooled SEM    12  0.6  1.5  0.05  2.2  Ca, %  0.55  8  1161  52.7  92.6  1.77  12.1  0.75  8  1169  53.1  91.5  1.74  2.1  0.95  8  1132  51.3  88.6  1.72  6.3  1.15  8  970  43.7  79.1  1.95  11.7  Pooled SEM    16  0.8  2.0  0.07  3.2  P-value  P    <0.0001  <0.0001  <0.0001  0.03  0.007    Ca    <0.0001  <0.0001  0.004  0.13  0.11    Ca × P    <0.0001  <0.0001  0.02  0.16  0.19  TP  Ca    Final BW,  ADG,  ADFI,  F/G,  Mortality,  level, %  level, %  Reps  g  g/d/bird  g/d/bird  g/g  %  0.60  0.55  4  1248a,b  56.9b,c  97.3a  1.71  10.0    0.75  4  1283a  58.5a,b  97.5a  1.67  0.0    0.95  4  1250a,b  56.7b,c  98.4a  1.73  2.5    1.15  4  1190b,c  54.2c  92.8a,b  1.72  0.8  0.45  0.55  4  1072d  48.5d  87.8b,c  1.82  14.2    0.75  4  1055d  47.7d  85.4c,d  1.81  4.2    0.95  4  1014d  45.7d  78.8d  1.72  10.0    1.15  4  750e  33.2e  65.4e  2.18  22.5  Pooled SEM    23  1.1  2.9  0.10  4.5  TP, %  0.60  16  1248  56.6  96.5  1.71b  3.3b  0.45  16  973  43.7  79.3  1.88a  12.7a  Pooled SEM    12  0.6  1.5  0.05  2.2  Ca, %  0.55  8  1161  52.7  92.6  1.77  12.1  0.75  8  1169  53.1  91.5  1.74  2.1  0.95  8  1132  51.3  88.6  1.72  6.3  1.15  8  970  43.7  79.1  1.95  11.7  Pooled SEM    16  0.8  2.0  0.07  3.2  P-value  P    <0.0001  <0.0001  <0.0001  0.03  0.007    Ca    <0.0001  <0.0001  0.004  0.13  0.11    Ca × P    <0.0001  <0.0001  0.02  0.16  0.19  a–eMeans with different superscripts within the same column differ (P < 0.05). TP = total phosphorus; Ca = calcium; Reps = replicates; BW = body weight; ADG = average daily weight gain; ADFI = average daily feed intake; F/G = feed/gain. View Large Serum Characteristics Serum Ca concentration was affected by dietary P (P < 0.05) and the interactions (P < 0.05), but not by dietary Ca (P > 0.05). The P concentration (P < 0.05) and ALP activity (P < 0.05) in serum of ducklings at d 21 were affected by dietary P, dietary Ca, and their interactions (Table 3). Compared to the normal-P group, the low-P group had decreased concentrations of Ca and P and increased ALP activity and CT concentration in the serum of ducklings at d 21. In the normal-P group, ducklings fed the medium-Ca diet had higher (P < 0.05) serum Ca concentrations than birds fed the other 3 diets, while no differences (P > 0.05) were observed in serum P concentration and ALP activity of ducklings among dietary Ca treatments. In the low-P group, serum Ca and P concentrations were decreased (P < 0.05) as dietary Ca level increased from 0.55 to 0.95%, but had an increase (P < 0.05) at 1.15%, while serum ALP activity was increased (P < 0.05) as dietary Ca level increased from 0.55 to 1.15%. Table 3. Effect of dietary Ca level on serum characteristic of ducklings subjected to the normal- and low-P diets at d 21 in Exp. 1. TP level, %  Ca level, %  Reps  Ca, mmol/L  P, mmol/L  CT, pg/mL  ALP, U/L  0.60  0.55  4  1.74b,c  2.96a  39.8  38.2d    0.75  4  1.79b,c  2.91a  39.7  47.7c,d    0.95  4  2.49a  2.71a,b  41.2  40.6c,d    1.15  4  1.90b  2.80a,b  44.5  38.7d  0.45  0.55  4  1.85b,c  2.92a  56.3  57.0c  0.75  4  1.44c,d  2.13c  62.5  74.8b  0.95  4  1.33d  1.72e  43.5  85.7a,b  1.15  4  1.85b,c  2.19c,d  58.4  93.0a  Pooled SEM    0.14  0.12  5.8  6.0  TP, %  0.60  16  1.97  2.85  41.3b  41.3    0.45  16  1.62  2.24  55.2a  77.6  Pooled SEM    0.07  0.06  3.0  3.1  Ca, %  0.55  8  1.79  2.94  48.0  47.6    0.75  8  1.60  2.52  51.1  61.3    0.95  8  1.91  2.22  42.3  63.2    1.15  8  1.88  2.50  51.5  65.9  Pooled SEM    0.10  0.09  4.2  4.3  P-value  P    0.001  <0.0001  0.001  <0.0001    Ca    0.16  <0.0001  0.44  0.02    Ca × P    0.0003  0.002  0.43  0.02  TP level, %  Ca level, %  Reps  Ca, mmol/L  P, mmol/L  CT, pg/mL  ALP, U/L  0.60  0.55  4  1.74b,c  2.96a  39.8  38.2d    0.75  4  1.79b,c  2.91a  39.7  47.7c,d    0.95  4  2.49a  2.71a,b  41.2  40.6c,d    1.15  4  1.90b  2.80a,b  44.5  38.7d  0.45  0.55  4  1.85b,c  2.92a  56.3  57.0c  0.75  4  1.44c,d  2.13c  62.5  74.8b  0.95  4  1.33d  1.72e  43.5  85.7a,b  1.15  4  1.85b,c  2.19c,d  58.4  93.0a  Pooled SEM    0.14  0.12  5.8  6.0  TP, %  0.60  16  1.97  2.85  41.3b  41.3    0.45  16  1.62  2.24  55.2a  77.6  Pooled SEM    0.07  0.06  3.0  3.1  Ca, %  0.55  8  1.79  2.94  48.0  47.6    0.75  8  1.60  2.52  51.1  61.3    0.95  8  1.91  2.22  42.3  63.2    1.15  8  1.88  2.50  51.5  65.9  Pooled SEM    0.10  0.09  4.2  4.3  P-value  P    0.001  <0.0001  0.001  <0.0001    Ca    0.16  <0.0001  0.44  0.02    Ca × P    0.0003  0.002  0.43  0.02  a–eMeans with different superscripts within the same column differ (P < 0.05). TP = total phosphorus; Ca = calcium; Reps = replicates; CT = calcitonin; ALP = alkaline phosphatase. View Large Tibia Bone Characteristics The bone ash content (P < 0.05) and BMD (P < 0.05) of ducklings at d 21 were affected by dietary P, dietary Ca, and their interactions. Bone Ca and P contents were affected (P < 0.05) by dietary P, but not by dietary Ca (P > 0.05) nor by their interactions (P > 0.05) (Table 4). Ducklings fed the low-P diet had lower contents of ash, Ca, and P in bone and BMD than birds fed the normal-P diet. In the normal-P group, bone ash content was increased (P < 0.05) as dietary Ca content increased from 0.55 to 1.15%, while BMD of ducklings was not affected (P > 0.05) by dietary Ca levels. When subjected to the low-P diet, ducklings from the high-Ca group had lower (P < 0.05) bone ash content and BMD than the other 3 dietary Ca groups. Table 4. Effect of dietary Ca level on tibia bone characteristic of ducklings subjected to the normal- and low-P diets at d 21 in Exp. 1. TP level, %  Ca level, %  Reps  Ash, %  Ca, %  P, %  BMD, mg/mm−2  0.60  0.55  4  36.9c  34.8  17.2  166a    0.75  4  40.0b  34.6  17.3  180a    0.95  4  41.3a,b  35.4  17.2  180a    1.15  4  40.4b  35.5  17.0  171a  0.45  0.55  4  37.1c  34.3  16.3  121b    0.75  4  37.2c  34.7  15.9  118b    0.95  4  37.6c  34.3  15.3  115b    1.15  4  31.0d  34.4  15.3  94c  Pooled SEM    0.90  0.40  0.22  5.2  TP, %  0.60  16  39.6  35.1a  17.2a  174  0.45  16  35.7  34.4b  15.7b  112  Pooled SEM    0.44  0.20  0.11  2.6  Ca, %  0.55  8  37.0  34.5  16.7  143    0.75  8  38.6  34.6  16.6  149    0.95  8  39.4  34.8  16.3  148    1.15  8  35.7  34.9  16.2  132  Pooled SEM    0.62  0.28  0.15  3.7  P-value  P    <0.0001  0.03  <0.0001  <0.0001  Ca    0.01  0.73  0.07  0.01  Ca × P    <0.0001  0.46  0.14  0.04  TP level, %  Ca level, %  Reps  Ash, %  Ca, %  P, %  BMD, mg/mm−2  0.60  0.55  4  36.9c  34.8  17.2  166a    0.75  4  40.0b  34.6  17.3  180a    0.95  4  41.3a,b  35.4  17.2  180a    1.15  4  40.4b  35.5  17.0  171a  0.45  0.55  4  37.1c  34.3  16.3  121b    0.75  4  37.2c  34.7  15.9  118b    0.95  4  37.6c  34.3  15.3  115b    1.15  4  31.0d  34.4  15.3  94c  Pooled SEM    0.90  0.40  0.22  5.2  TP, %  0.60  16  39.6  35.1a  17.2a  174  0.45  16  35.7  34.4b  15.7b  112  Pooled SEM    0.44  0.20  0.11  2.6  Ca, %  0.55  8  37.0  34.5  16.7  143    0.75  8  38.6  34.6  16.6  149    0.95  8  39.4  34.8  16.3  148    1.15  8  35.7  34.9  16.2  132  Pooled SEM    0.62  0.28  0.15  3.7  P-value  P    <0.0001  0.03  <0.0001  <0.0001  Ca    0.01  0.73  0.07  0.01  Ca × P    <0.0001  0.46  0.14  0.04  a–dMeans with different superscripts within the same column differ (P < 0.05). TP = total phosphorus; Ca = calcium; Reps = replicates; BMD = bone mineral density. View Large Nutrient Utilization Dietary Ca had an effect (P < 0.05) on the utilization of energy, Ca, and P, but had no effect (P > 0.05) on the CP utilization (Table 5). Dietary P affected (P < 0.05) the utilization of CP and Ca, but did not affect (P > 0.05) energy and P utilization. There were significant interactions (P < 0.05) between dietary P and dietary Ca on the utilization of energy and Ca. Low-P diet decreased the ducks’ utilization of Ca and CP compared to normal-P diet. High-Ca diet decreased (P < 0.05) the utilization of Ca and P more than the other 3 dietary Ca levels. Under the normal-P diet, energy utilization was not affected (P > 0.05) by dietary Ca levels. Under the low-P diet, energy utilization was decreased (P < 0.05) in high-Ca diet compared to the low-Ca diet, with no difference among high-Ca, normal-Ca, and medium-Ca diets. Under the normal-P and low-P diets, Ca utilization was decreased (P < 0.05) as dietary Ca level increased. Especially, Ca utilization in both high-Ca and medium-Ca diets under the low-P diet was much lower (P < 0.05) than that in the diets with the same Ca levels under normal-P diet, respectively. Table 5. Effect of dietary Ca level on nutrient utilization of ducks subjected to the normal- and low-P diets in Exp. 2. TP level, %  Ca level, %  Reps  energy, %  CP, %  Ca, %  P, %  0.60  0.55  6  76.5a,b  54.1  38.4a  33.9    0.75  6  75.9a,b  51.5  33.4b,c  34.8    0.95  6  77.6a  53.4  32.8b,c  37.3    1.15  6  76.5a,b  52.6  25.6d  26.4  0.45  0.55  6  78.0a  51.4  33.3b,c  37.2    0.75  6  76.6a,b  48.7  31.1c  35.6    0.95  6  76.3a,b  51.9  23.7d  34.8    1.15  6  74.8b  48.3  15.1e  31.9  Pooled SEM    0.57  1.42  1.30  1.67  TP, %  0.60  24  76.6  52.9a  32.5  33.1  0.45  24  76.4  50.0b  25.7  34.9  Pooled SEM    0.28  0.71  0.67  0.84  Ca, %  0.55  12  77.2  52.8  32.5  35.5a    0.75  12  76.2  50.1  30.3  35.2a    0.95  12  76.9  52.6  28.2  36.1a    1.15  12  75.6  50.5  20.3  29.1b  Pooled SEM    0.40  1.00  0.94  1.18  P-value  P    0.65  0.007  <0.0001  0.14  Ca    0.03  0.14  <0.0001  0.0005  Ca × P    0.02  0.81  0.01  0.11  TP level, %  Ca level, %  Reps  energy, %  CP, %  Ca, %  P, %  0.60  0.55  6  76.5a,b  54.1  38.4a  33.9    0.75  6  75.9a,b  51.5  33.4b,c  34.8    0.95  6  77.6a  53.4  32.8b,c  37.3    1.15  6  76.5a,b  52.6  25.6d  26.4  0.45  0.55  6  78.0a  51.4  33.3b,c  37.2    0.75  6  76.6a,b  48.7  31.1c  35.6    0.95  6  76.3a,b  51.9  23.7d  34.8    1.15  6  74.8b  48.3  15.1e  31.9  Pooled SEM    0.57  1.42  1.30  1.67  TP, %  0.60  24  76.6  52.9a  32.5  33.1  0.45  24  76.4  50.0b  25.7  34.9  Pooled SEM    0.28  0.71  0.67  0.84  Ca, %  0.55  12  77.2  52.8  32.5  35.5a    0.75  12  76.2  50.1  30.3  35.2a    0.95  12  76.9  52.6  28.2  36.1a    1.15  12  75.6  50.5  20.3  29.1b  Pooled SEM    0.40  1.00  0.94  1.18  P-value  P    0.65  0.007  <0.0001  0.14  Ca    0.03  0.14  <0.0001  0.0005  Ca × P    0.02  0.81  0.01  0.11  a–eMeans with different superscripts within the same column differ (P < 0.05). TP = total phosphorus; Ca = calcium; Reps = replicates; CP = crude protein. View Large DISCUSSION The depression of the growth performance and high mortality due to dietary P deficiency has been well demonstrated in the previous studies about P requirement in poultry (Waldroup et al., 2000; Yan et al., 2001; Liu, et al., 2017). In the present study, similar results were found in ducklings fed a low-P diet below the recommendation of NRC (1994) during the starter period. Some studies showed that high Ca levels had negative effects on growth performance in broilers that received diets with normal P levels (Rao et al., 2006; Hamdi et al., 2015), which was confirmed in ducklings in the current study. When under the low-P group, final BW, ADG, and ADFI were decreased as dietary Ca level increased from 0.55 to 1.15%. In addition, we found that high-Ca diet induced greater growth suppression in ducklings subjected to a low-P diet compared to the other 3 lower Ca diets. The magnitude of the depression may be due to the improper Ca to P ratios (Hamdi et al., 2015), because higher Ca to P ratios in diets tend to form the insoluble Ca-P complexes, resulting in reduced absorption of Ca and P (Simpson and Wise, 1990). However, the adverse effects of dietary high Ca to P ratios on growth performance were not observed in large domestic animals, such as cattle (Dowe et al., 1957) and gilts (Liptrap et al., 1970). It was suggested that poultry, with a shorter growth period, have greater sensitivity to dietary Ca excess. Therefore, the potential negative effect of higher Ca to P ratio in diets should be carefully considered when feeding diets with higher Ca level than the recommendation of NRC (1994), which were adopted for fast-growing strains to prevent skeletal problems and leg abnormalities in ducklings (Roberson et al., 2004). Faridi et al. (2015) has demonstrated that the negative effect of high Ca levels could be alleviated by increasing P content or improving P utilization (supplementation of phytase and vitamin D3) in diets. Serum Ca and P concentrations within a narrow physiological range are sensitive to reflect the body Ca and P metabolism (Veum, 2010). In the current study, the highest and lowest serum Ca concentrations were obtained in ducklings fed the diet with 0.95% Ca level under the normal- and low-P diets, respectively. The opposite results indicated that Ca and P ratio was a predominant factor in affecting Ca utilization. Under the normal-P diet, dietary Ca levels did not affect serum P concentration. However, under the low-P diet, serum P concentration was decreased as the dietary Ca increased from 0.55 to 0.95%, suggesting that increasing Ca level in the diet might inhibit P utilization and induced a greater P deficiency in ducks fed the low-P diet. The ALP enzyme activity, which is localized in the plasma membrane of osteoblasts before extracellular release, correlates with bone reabsorption (Golub and Boesze-Battaglia, 2007). The present study found that serum ALP activity was increased as dietary Ca level increased, indicating that ducklings might promote bone reabsorption to obtain P for important metabolism activities (energy metabolism, cellular signaling, membrane integrity, enzyme activity, etc.) when dietary P concentration is below the requirement. The highest serum ALP activity associated with the enhanced bone reabsorption might explain why serum P was increased at the high-Ca group compared to the medium-Ca group with a lower P diet. Calcitonin is a hormone known to participate in the regulation of Ca and P metabolism (Copp et al., 1962). In rats, P deprivation has been demonstrated to be a potent stimulus to serum CT secretion (Catherwood et al., 1983). In the current study, an elevated serum CT concentration was observed in ducklings fed a low-P diet. High CT concentration can inhibit osteoclast activity in bones (Yamamoto et al., 2005) and increase excretion of Ca and P in urinary (Carney, 1997), thus leading to the decreased serum Ca and P concentrations in the P-deficient ducklings. Bone mineralization was also influenced by dietary Ca and P in human and animals (Cumming, 1990; Hamdi et al., 2015). Similarly, in ducks, the low-P group decreased bone ash content and BMD compared to the normal-P group in our study. In broilers, the added Ca level from 0.40 to 0.90% in diets increased tibia bone ash content (Driver et al., 2005). However, high dietary Ca levels above the Ca requirement induced bone mineralization losses in broilers (Rao et al., 2006; Hamdi et al., 2015). In the present study, under the normal P diet, bone ash content was increased as dietary Ca content increased from 0.55 to 0.95%, but was decreased when at the highest Ca level with 1.15%. This effect might be explained by the reduction in Ca and P absorption associated with high Ca levels for bone mineralization (Liem et al., 2009). When ducklings from the low-P group were given a high-Ca diet, it further induced greater losses of bone ash content and BMD. These results agreed with those in the previous studies of broilers (Lei, et al., 1994; Sebastian et al., 1996). On the one hand, due to excess Ca in diets, the P utilization for bone mineralization was reduced by the formation of an insoluble complex in gut (Plumstead et al., 2008). On the other hand, more bone P was reabsorbed to maintain important metabolic actions when birds were fed high-Ca diets in P-deficient state (Cuisnier-Gleizes et al., 1976). In Exp. 2, we further evaluate the the utilization of energy, CP, Ca, and P of 8 diets used in Exp. 1 using the Sibbald method as described by Adeola et al. (1997). In the current study, low-P diet decreased the utilization of CP and Ca compared to normal-P diet, which may contribute to a poor feed conversion ratio observed in the ducks fed a low-P diet. In addition, a significant decrease in the utilization of Ca and P were observed in ducks fed high-Ca diet. As reported previously, a high dietary Ca level increased the intestinal pH and then decreased the apparent digestibility of Ca and P in broilers (Sebastian et al., 1996; Adeola and Walk, 2013) by the formation of insoluble Ca-P complexes (Plumstead et al., 2008). Under low P diet, the decline of Ca utilization at high-Ca diet became more aggravated, which might contribute to the greater loss of bone mineralization. In addition, under the low P diet, high-Ca diet decreased energy utilization of birds. Some studies showed that excess Ca was able to form insoluble soaps with free fatty acids and bile acids and these soaps lowered the utilization of energy derived from lipids, particularly saturated fats, in vivo (Gacs and Barltrop, 1977; Govers et al., 1996; Shahkhalili et al., 2001). In conclusion, dietary low-P level decreased growth performance and reduced serum Ca and P levels, bone ash content in bone and BMD in ducklings. High dietary Ca level induced a greater growth suppression and bone mineralization loss in ducklings fed a low-P diet. The aggravated negative effect of high dietary Ca level at a low-P level might be related to the reduced utilization of energy, Ca and P, and the elevated serum ALP activity. Acknowledgements This study was sponsored by National Waterfowl Industry Program in China CARS-42–15) and Special Fund for Agro-scientific Research in the Public Interest (201303143). REFERENCES Adeola O., Walk C. L.. 2013. 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High calcium to phosphorus ratio impairs growth and bone mineralization in Pekin ducklings

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© 2018 Poultry Science Association Inc.
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0032-5791
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Abstract

Abstract Two experiments were conducted to investigate the effect of high dietary calcium (Ca) level on growth performance, Ca and phosphorus (P) metabolism, and nutrient utilization in ducklings subjected to normal and low P levels in diets. A completely randomized design was used with a factorial arrangement of 2 total dietary P levels [normal-P (0.60%) and low-P (0.45%) groups] × 4 dietary Ca levels [low-Ca (0.55%), normal-Ca (0.75%), medium-Ca (0.95%) and high-Ca (1.15%) groups)]. Compared to normal-P group, low-P group had lower (P < 0.05) final body weight (BW), average daily gain (ADG), and average daily feed intake (ADFI) and reduced (P < 0.05) serum Ca and P levels, bone Ca, P, and ash content, and bone mineral density in ducklings during the starter period. Under the low-P group, birds from high-Ca group had lower (P < 0.05) final BW, ADG, ADFI, bone ash content, bone mineral density, and the utilization of energy, Ca, and P than those from low-Ca, normal-Ca, and medium-Ca groups. Our results indicate that high-Ca diet induced greater growth suppression and bone mineralization loss in ducklings fed a low-P diet. The aggravated negative effect of high dietary Ca level with a low P level might be related to the elevated serum alkaline phosphatase activity and the reduced utilization of energy, Ca, and P. INTRODUCTION Calcium (Ca) is an essential mineral nutrient for optimal growth and bone development in poultry (Atteh and Leeson, 1984). The growth of modern meat-breed ducks has been accelerated during recent decades (Farrell, 1990). The NRC (1994) recommendation of Ca (0.60∼0.65%) for ducks, based on a 1967 study (Dean et al., 1967), may not be applicable for modern ducks. In practical production, a tendency to over-supplement (0.85∼1.10%) Ca as an insurance factor to prevent skeletal problems and leg abnormalities has been adopted for fast-growing strains of ducks (Applegate and Angel, 2014; Roberson et al., 2004). Previous studies in broilers showed that daily weight gain and bone ash content were increased as dietary Ca content increased from 0.40 to 0.90% (Driver et al., 2005). However, when dietary Ca levels are above the Ca requirement, it might impair growth performance and decrease bone mineralization in broilers (Rao et al., 2006; Hamdi et al., 2015). There must be recognition of the possible adverse effects of high Ca intake on the metabolism of other mineral nutrients (Schiller et al., 1989; Wood and Zheng, 1997; Minihane and Fairweather-Tait, 1998). High Ca intake induced a significant reduction in the efficiency of phosphorus (P) absorption in poultry (Simpson and Wise, 1990). Excess Ca can interact with inorganic P to form the insoluble complexes (Schiller et al., 1989), which reduced the utilization of Ca and P (Plumstead et al., 2008). So far, the negative effects of high dietary Ca on duck performance have not been well demonstrated. It is assumed that the deleterious effect of high Ca intake would be even more evident at limited amounts of available P or P deficiency in the diets. Therefore, the objective of this study was to investigate the effect of high dietary Ca level on the growth performance, characteristics of serum and tibia bone, and nutrient utilization in ducklings subjected to the normal-P and low-P level diets. MATERIALS AND METHODS Animals and Diets All experimental procedures were approved by the Institutional Animal Care and Use Committee of South China Agricultural University. The study included feeding (Exp. 1) and metabolic experiments with ducks (Exp. 2). In Exp. 1, 960 day-old male Cherry Valley ducklings were weighed individually and allotted to 8 dietary treatments. Each treatment was 4 replicated pen with 30 ducklings per pen. Feed and water were provided ad libitum from hatch to d 21 during the experimental period. The experimental design was completely randomized with a 2 × 4 factorial arrangement of treatments. These variables included 2 total dietary P levels at 0.60% (normal-P group) and 0.45% (low-P group) and 4 dietary Ca levels at 0.55% (low-Ca group), 0.75% (normal-Ca group), 0.95% (medium-Ca group), 1.15% (high-Ca group). The basal diets were formulated to meet or exceed nutrition requirements recommended by NRC (1994) for ducklings at the starter period except Ca and P. Composition and nutrient levels of the experimental diets were presented in Table 1. At 21 d of age, after 12 h feed withdrawal, birds were weighed and feed consumption and mortality were recorded by each replicate pen. The average daily gain (ADG), average daily feed intake (ADFI) and feed/gain ratio (F/G) were calculated. Based on the average body weight (BW) of birds in each replicate pen, 2 birds in each pen were taken for blood sampling, and then were euthanized by CO2 inhalation, and the tibias were removed and used for measuring the indices related to bone characteristics. In Exp. 2, the Sibbald method described by McNab and Blair (1988) was used to evaluate the utilization of energy, CP, Ca, and P of 8 diets used in Exp. 1 for ducks. Briefly, 6 male Cherry Valley ducks, age 10 wk, of similar weight (3.7 to 3.8 kg/bird) were assigned to each of 8 test diets. As described previously (Adeola et al., 1997; King et al., 1997), during the adaptation period of the first 48 h, all the birds were intubated with dextrose solution (30 g/100 mL of water) at 8 and 32 h after feed was removed. Thirty grams (30 g/100 mL of water) of each diet were tube-fed to each bird at 48 h after feed was withdrawn. In order to reduce excessive weight loss and the variability in endogenous nutrient loss (ENL), another 6 ducks that served as controls for estimation of endogenous losses of Ca, P, CP and energy were tube-fed 30 g dextrose (30 g/100 mL of water) at 48 h after feed was withdrawn (Adeola et al., 1997). Total excreta samples were collected during the next 48 h from each bird using Playtex bottles with attached Whirl-pak bags (McNab and Blair, 1988). As described by Adeola et al. (1997), the standardized values of nutrient utilization (NU) in the experimental diets were calculated using the following formula: NU = (NI–NO + ENL)/NI × 100%, where NI = nutrient intake of the diet; NO = nutrient output of the excreta; ENL = endogenous nutrient loss. Table 1. Composition and nutrient levels of the experimental diets (as-fed basis). Total P level, %  0.60  0.45  Ca level, %  0.55  0.75  0.95  1.15  0.55  0.75  0.95  1.15  Corn  63.60  63.40  63.08  63.00  63.65  63.60  63.43  63.01  Soybean meal  23.5  22.5  21.5  22.0  24.0  24.0  23.0  23.5  Corn gluten meal  5.60  6.25  7.00  7.80  4.70  5.40  6.50  7.00  Cottonseed meal  2.00  2.00  2.00  1.26  2.30  1.60  1.10  1.00  Rapeseed meal  2.00  2.00  2.00  1.00  2.50  2.00  2.00  1.00  L-lysine·HCl  0.40  0.42  0.45  0.45  0.39  0.39  0.42  0.42  DL-Methionine  0.15  0.15  0.15  0.14  0.16  0.16  0.15  0.15  Dicalcium phosphate  1.65  1.65  1.65  1.65  0.70  0.75  0.75  0.75  Sodium chloride  0.30  0.30  0.30  0.30  0.30  0.30  0.30  0.30  Limestone  0.30  0.83  1.37  1.90  0.80  1.30  1.85  2.37  Vitamin and mineral premix1  0.50  0.50  0.50  0.50  0.50  0.50  0.50  0.50  Total  100  100  100  100  100  100  100  100  Nutrient composition                  Calculated value, %                  ME, kcal/kg  2895  2895  2895  2895  2895  2895  2895  2895  Crude protein  20.5  20.5  20.5  20.5  20.5  20.5  20.5  20.5  Lysine  1.17  1.17  1.17  1.17  1.17  1.17  1.17  1.17  Methionine  0.48  0.48  0.48  0.48  0.48  0.48  0.48  0.48  Methionine + cysteine  0.81  0.81  0.81  0.81  0.81  0.81  0.81  0.81  Calcium  0.55  0.75  0.95  1.15  0.55  0.75  0.95  1.15  Total phosphorus  0.60  0.60  0.60  0.60  0.45  0.45  0.45  0.45  Analyzed value2, %                  Calcium  0.60  0.78  0.95  1.15  0.63  0.78  0.93  1.20  Total phosphorus  0.57  0.61  0.61  0.60  0.45  0.50  0.49  0.47  Total P level, %  0.60  0.45  Ca level, %  0.55  0.75  0.95  1.15  0.55  0.75  0.95  1.15  Corn  63.60  63.40  63.08  63.00  63.65  63.60  63.43  63.01  Soybean meal  23.5  22.5  21.5  22.0  24.0  24.0  23.0  23.5  Corn gluten meal  5.60  6.25  7.00  7.80  4.70  5.40  6.50  7.00  Cottonseed meal  2.00  2.00  2.00  1.26  2.30  1.60  1.10  1.00  Rapeseed meal  2.00  2.00  2.00  1.00  2.50  2.00  2.00  1.00  L-lysine·HCl  0.40  0.42  0.45  0.45  0.39  0.39  0.42  0.42  DL-Methionine  0.15  0.15  0.15  0.14  0.16  0.16  0.15  0.15  Dicalcium phosphate  1.65  1.65  1.65  1.65  0.70  0.75  0.75  0.75  Sodium chloride  0.30  0.30  0.30  0.30  0.30  0.30  0.30  0.30  Limestone  0.30  0.83  1.37  1.90  0.80  1.30  1.85  2.37  Vitamin and mineral premix1  0.50  0.50  0.50  0.50  0.50  0.50  0.50  0.50  Total  100  100  100  100  100  100  100  100  Nutrient composition                  Calculated value, %                  ME, kcal/kg  2895  2895  2895  2895  2895  2895  2895  2895  Crude protein  20.5  20.5  20.5  20.5  20.5  20.5  20.5  20.5  Lysine  1.17  1.17  1.17  1.17  1.17  1.17  1.17  1.17  Methionine  0.48  0.48  0.48  0.48  0.48  0.48  0.48  0.48  Methionine + cysteine  0.81  0.81  0.81  0.81  0.81  0.81  0.81  0.81  Calcium  0.55  0.75  0.95  1.15  0.55  0.75  0.95  1.15  Total phosphorus  0.60  0.60  0.60  0.60  0.45  0.45  0.45  0.45  Analyzed value2, %                  Calcium  0.60  0.78  0.95  1.15  0.63  0.78  0.93  1.20  Total phosphorus  0.57  0.61  0.61  0.60  0.45  0.50  0.49  0.47  1Provided per kilogram of diet: vitamin A, 4,000 IU; vitamin D3, 2,000 IU; vitamin E, 24 IU; thiamine, 2.0 mg; riboflavin, 12 mg; pyridoxine, 4.0 mg; vitamin B12, 0.02 mg; calcium pantothenate, 10 mg; folate, 0.15 mg; niacin, 50 mg; biotin, 0.15 mg; choline (Choline chloride), 1,000 mg; Cu (CuSO4·5H2O), 8 mg; Fe (FeSO4·7H2O), 80 mg; Zn (ZnSO4·7H2O), 90 mg; Mn (MnSO4·H2O), 70 mg; Se (NaSeO3), 0.3 mg; I (KI), 0.4 mg. 2Analysed values based on triplicate determinations. View Large Sample Collections and Preparations Blood samples were obtained via a bronchial vein (3.5 mL/bird), immediately placed on ice, and then centrifuged at 2,000 × g for 15 min in a refrigerated centrifuge to prepare serum. The serum samples were stored at −20°C for the analysis of Ca and P concentrations and other biochemical parameters. Tibia bones were removed and then were dried at 105°C for 24 h, and then defatted 48 h in ethyl alcohol followed by a 48 h extraction in ethyl ether, and then dried for 12 h at 110°C. Tibia bone ash percentage were determined by ashing overnight at 550°C with a muffle furnace. Diets, bone ash, and freeze-dried excreta samples were analyzed for crude protein (CP) (method 988.05; Association of Official Analytical Chemists (AOAC), 1990), and Ca and total P (method 985.01; AOAC, 1990). The bone mineral density (BMD) were measured by DEXA (DCS-600; Aloka, Tokyo, Japan) as described by Liu et al. (2017). Gross energy was determined using an adiabatic calorimetric bomb (IKA C200; Heitersheim, Germany). The Ca and P concentrations and alkaline phosphatase (ALP) activity in serum was determined by HITACHI 7180 automatic biochemical analyzer (Hitachi Ltd., Tokyo, Japan) with commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Serum calcitonin (CT) was measured by radioimmunoassay using commercial kits (Beijing North Institute of Biological Technology, Beijing, China). Statistical Analyses All data were analyzed by 2-way analysis of variance (ANOVA) using the general linear model procedure of SAS 9.2 (SAS Institute, 2010). The model included the main effects of dietary P, dietary Ca, and their interactions. Each replicate cage (Exp.1) or duck (Exp.2) served as an experimental unit. Data from mortality of ducklings were transformed to arcsin before statistical analysis. Differences among means were tested by the Least Square Difference method, and statistical significance was set at P < 0.05. RESULTS Growth Performance Dietary P, dietary Ca, and their interactions affect the final BW (P < 0.0001), ADG (P < 0.0001) and ADFI (P < 0.05) of ducklings from hatch to d 21 (Table 2). The F/G and mortality was affected (P < 0.05) by dietary P, but not by dietary Ca (P > 0.05) and their interactions (P > 0.05; Table 1). The high-Ca diet decreased (P < 0.05) the final BW, ADG, and ADFI of ducklings from hatch to d 21 compared to the other 3 Ca diets. Compared to normal-P group, low-P group decreased the final BW, ADG, and ADFI, and increased F/G and mortality of ducklings from hatch to d 21. Under the normal-P, no differences (P > 0.05) were observed in the final BW, ADG, and ADFI of ducklings as dietary Ca increased from 0.55 to 0.95%; under the low-P group, however, final BW, ADG, and ADFI were decreased (P < 0.05) as dietary Ca level increased from 0.55 to 1.15%. Table 2. Effect of dietary Ca level on growth performance and mortality of ducklings subjected to the normal and low P diets from hatch to d 21 in Exp. 1. TP  Ca    Final BW,  ADG,  ADFI,  F/G,  Mortality,  level, %  level, %  Reps  g  g/d/bird  g/d/bird  g/g  %  0.60  0.55  4  1248a,b  56.9b,c  97.3a  1.71  10.0    0.75  4  1283a  58.5a,b  97.5a  1.67  0.0    0.95  4  1250a,b  56.7b,c  98.4a  1.73  2.5    1.15  4  1190b,c  54.2c  92.8a,b  1.72  0.8  0.45  0.55  4  1072d  48.5d  87.8b,c  1.82  14.2    0.75  4  1055d  47.7d  85.4c,d  1.81  4.2    0.95  4  1014d  45.7d  78.8d  1.72  10.0    1.15  4  750e  33.2e  65.4e  2.18  22.5  Pooled SEM    23  1.1  2.9  0.10  4.5  TP, %  0.60  16  1248  56.6  96.5  1.71b  3.3b  0.45  16  973  43.7  79.3  1.88a  12.7a  Pooled SEM    12  0.6  1.5  0.05  2.2  Ca, %  0.55  8  1161  52.7  92.6  1.77  12.1  0.75  8  1169  53.1  91.5  1.74  2.1  0.95  8  1132  51.3  88.6  1.72  6.3  1.15  8  970  43.7  79.1  1.95  11.7  Pooled SEM    16  0.8  2.0  0.07  3.2  P-value  P    <0.0001  <0.0001  <0.0001  0.03  0.007    Ca    <0.0001  <0.0001  0.004  0.13  0.11    Ca × P    <0.0001  <0.0001  0.02  0.16  0.19  TP  Ca    Final BW,  ADG,  ADFI,  F/G,  Mortality,  level, %  level, %  Reps  g  g/d/bird  g/d/bird  g/g  %  0.60  0.55  4  1248a,b  56.9b,c  97.3a  1.71  10.0    0.75  4  1283a  58.5a,b  97.5a  1.67  0.0    0.95  4  1250a,b  56.7b,c  98.4a  1.73  2.5    1.15  4  1190b,c  54.2c  92.8a,b  1.72  0.8  0.45  0.55  4  1072d  48.5d  87.8b,c  1.82  14.2    0.75  4  1055d  47.7d  85.4c,d  1.81  4.2    0.95  4  1014d  45.7d  78.8d  1.72  10.0    1.15  4  750e  33.2e  65.4e  2.18  22.5  Pooled SEM    23  1.1  2.9  0.10  4.5  TP, %  0.60  16  1248  56.6  96.5  1.71b  3.3b  0.45  16  973  43.7  79.3  1.88a  12.7a  Pooled SEM    12  0.6  1.5  0.05  2.2  Ca, %  0.55  8  1161  52.7  92.6  1.77  12.1  0.75  8  1169  53.1  91.5  1.74  2.1  0.95  8  1132  51.3  88.6  1.72  6.3  1.15  8  970  43.7  79.1  1.95  11.7  Pooled SEM    16  0.8  2.0  0.07  3.2  P-value  P    <0.0001  <0.0001  <0.0001  0.03  0.007    Ca    <0.0001  <0.0001  0.004  0.13  0.11    Ca × P    <0.0001  <0.0001  0.02  0.16  0.19  a–eMeans with different superscripts within the same column differ (P < 0.05). TP = total phosphorus; Ca = calcium; Reps = replicates; BW = body weight; ADG = average daily weight gain; ADFI = average daily feed intake; F/G = feed/gain. View Large Serum Characteristics Serum Ca concentration was affected by dietary P (P < 0.05) and the interactions (P < 0.05), but not by dietary Ca (P > 0.05). The P concentration (P < 0.05) and ALP activity (P < 0.05) in serum of ducklings at d 21 were affected by dietary P, dietary Ca, and their interactions (Table 3). Compared to the normal-P group, the low-P group had decreased concentrations of Ca and P and increased ALP activity and CT concentration in the serum of ducklings at d 21. In the normal-P group, ducklings fed the medium-Ca diet had higher (P < 0.05) serum Ca concentrations than birds fed the other 3 diets, while no differences (P > 0.05) were observed in serum P concentration and ALP activity of ducklings among dietary Ca treatments. In the low-P group, serum Ca and P concentrations were decreased (P < 0.05) as dietary Ca level increased from 0.55 to 0.95%, but had an increase (P < 0.05) at 1.15%, while serum ALP activity was increased (P < 0.05) as dietary Ca level increased from 0.55 to 1.15%. Table 3. Effect of dietary Ca level on serum characteristic of ducklings subjected to the normal- and low-P diets at d 21 in Exp. 1. TP level, %  Ca level, %  Reps  Ca, mmol/L  P, mmol/L  CT, pg/mL  ALP, U/L  0.60  0.55  4  1.74b,c  2.96a  39.8  38.2d    0.75  4  1.79b,c  2.91a  39.7  47.7c,d    0.95  4  2.49a  2.71a,b  41.2  40.6c,d    1.15  4  1.90b  2.80a,b  44.5  38.7d  0.45  0.55  4  1.85b,c  2.92a  56.3  57.0c  0.75  4  1.44c,d  2.13c  62.5  74.8b  0.95  4  1.33d  1.72e  43.5  85.7a,b  1.15  4  1.85b,c  2.19c,d  58.4  93.0a  Pooled SEM    0.14  0.12  5.8  6.0  TP, %  0.60  16  1.97  2.85  41.3b  41.3    0.45  16  1.62  2.24  55.2a  77.6  Pooled SEM    0.07  0.06  3.0  3.1  Ca, %  0.55  8  1.79  2.94  48.0  47.6    0.75  8  1.60  2.52  51.1  61.3    0.95  8  1.91  2.22  42.3  63.2    1.15  8  1.88  2.50  51.5  65.9  Pooled SEM    0.10  0.09  4.2  4.3  P-value  P    0.001  <0.0001  0.001  <0.0001    Ca    0.16  <0.0001  0.44  0.02    Ca × P    0.0003  0.002  0.43  0.02  TP level, %  Ca level, %  Reps  Ca, mmol/L  P, mmol/L  CT, pg/mL  ALP, U/L  0.60  0.55  4  1.74b,c  2.96a  39.8  38.2d    0.75  4  1.79b,c  2.91a  39.7  47.7c,d    0.95  4  2.49a  2.71a,b  41.2  40.6c,d    1.15  4  1.90b  2.80a,b  44.5  38.7d  0.45  0.55  4  1.85b,c  2.92a  56.3  57.0c  0.75  4  1.44c,d  2.13c  62.5  74.8b  0.95  4  1.33d  1.72e  43.5  85.7a,b  1.15  4  1.85b,c  2.19c,d  58.4  93.0a  Pooled SEM    0.14  0.12  5.8  6.0  TP, %  0.60  16  1.97  2.85  41.3b  41.3    0.45  16  1.62  2.24  55.2a  77.6  Pooled SEM    0.07  0.06  3.0  3.1  Ca, %  0.55  8  1.79  2.94  48.0  47.6    0.75  8  1.60  2.52  51.1  61.3    0.95  8  1.91  2.22  42.3  63.2    1.15  8  1.88  2.50  51.5  65.9  Pooled SEM    0.10  0.09  4.2  4.3  P-value  P    0.001  <0.0001  0.001  <0.0001    Ca    0.16  <0.0001  0.44  0.02    Ca × P    0.0003  0.002  0.43  0.02  a–eMeans with different superscripts within the same column differ (P < 0.05). TP = total phosphorus; Ca = calcium; Reps = replicates; CT = calcitonin; ALP = alkaline phosphatase. View Large Tibia Bone Characteristics The bone ash content (P < 0.05) and BMD (P < 0.05) of ducklings at d 21 were affected by dietary P, dietary Ca, and their interactions. Bone Ca and P contents were affected (P < 0.05) by dietary P, but not by dietary Ca (P > 0.05) nor by their interactions (P > 0.05) (Table 4). Ducklings fed the low-P diet had lower contents of ash, Ca, and P in bone and BMD than birds fed the normal-P diet. In the normal-P group, bone ash content was increased (P < 0.05) as dietary Ca content increased from 0.55 to 1.15%, while BMD of ducklings was not affected (P > 0.05) by dietary Ca levels. When subjected to the low-P diet, ducklings from the high-Ca group had lower (P < 0.05) bone ash content and BMD than the other 3 dietary Ca groups. Table 4. Effect of dietary Ca level on tibia bone characteristic of ducklings subjected to the normal- and low-P diets at d 21 in Exp. 1. TP level, %  Ca level, %  Reps  Ash, %  Ca, %  P, %  BMD, mg/mm−2  0.60  0.55  4  36.9c  34.8  17.2  166a    0.75  4  40.0b  34.6  17.3  180a    0.95  4  41.3a,b  35.4  17.2  180a    1.15  4  40.4b  35.5  17.0  171a  0.45  0.55  4  37.1c  34.3  16.3  121b    0.75  4  37.2c  34.7  15.9  118b    0.95  4  37.6c  34.3  15.3  115b    1.15  4  31.0d  34.4  15.3  94c  Pooled SEM    0.90  0.40  0.22  5.2  TP, %  0.60  16  39.6  35.1a  17.2a  174  0.45  16  35.7  34.4b  15.7b  112  Pooled SEM    0.44  0.20  0.11  2.6  Ca, %  0.55  8  37.0  34.5  16.7  143    0.75  8  38.6  34.6  16.6  149    0.95  8  39.4  34.8  16.3  148    1.15  8  35.7  34.9  16.2  132  Pooled SEM    0.62  0.28  0.15  3.7  P-value  P    <0.0001  0.03  <0.0001  <0.0001  Ca    0.01  0.73  0.07  0.01  Ca × P    <0.0001  0.46  0.14  0.04  TP level, %  Ca level, %  Reps  Ash, %  Ca, %  P, %  BMD, mg/mm−2  0.60  0.55  4  36.9c  34.8  17.2  166a    0.75  4  40.0b  34.6  17.3  180a    0.95  4  41.3a,b  35.4  17.2  180a    1.15  4  40.4b  35.5  17.0  171a  0.45  0.55  4  37.1c  34.3  16.3  121b    0.75  4  37.2c  34.7  15.9  118b    0.95  4  37.6c  34.3  15.3  115b    1.15  4  31.0d  34.4  15.3  94c  Pooled SEM    0.90  0.40  0.22  5.2  TP, %  0.60  16  39.6  35.1a  17.2a  174  0.45  16  35.7  34.4b  15.7b  112  Pooled SEM    0.44  0.20  0.11  2.6  Ca, %  0.55  8  37.0  34.5  16.7  143    0.75  8  38.6  34.6  16.6  149    0.95  8  39.4  34.8  16.3  148    1.15  8  35.7  34.9  16.2  132  Pooled SEM    0.62  0.28  0.15  3.7  P-value  P    <0.0001  0.03  <0.0001  <0.0001  Ca    0.01  0.73  0.07  0.01  Ca × P    <0.0001  0.46  0.14  0.04  a–dMeans with different superscripts within the same column differ (P < 0.05). TP = total phosphorus; Ca = calcium; Reps = replicates; BMD = bone mineral density. View Large Nutrient Utilization Dietary Ca had an effect (P < 0.05) on the utilization of energy, Ca, and P, but had no effect (P > 0.05) on the CP utilization (Table 5). Dietary P affected (P < 0.05) the utilization of CP and Ca, but did not affect (P > 0.05) energy and P utilization. There were significant interactions (P < 0.05) between dietary P and dietary Ca on the utilization of energy and Ca. Low-P diet decreased the ducks’ utilization of Ca and CP compared to normal-P diet. High-Ca diet decreased (P < 0.05) the utilization of Ca and P more than the other 3 dietary Ca levels. Under the normal-P diet, energy utilization was not affected (P > 0.05) by dietary Ca levels. Under the low-P diet, energy utilization was decreased (P < 0.05) in high-Ca diet compared to the low-Ca diet, with no difference among high-Ca, normal-Ca, and medium-Ca diets. Under the normal-P and low-P diets, Ca utilization was decreased (P < 0.05) as dietary Ca level increased. Especially, Ca utilization in both high-Ca and medium-Ca diets under the low-P diet was much lower (P < 0.05) than that in the diets with the same Ca levels under normal-P diet, respectively. Table 5. Effect of dietary Ca level on nutrient utilization of ducks subjected to the normal- and low-P diets in Exp. 2. TP level, %  Ca level, %  Reps  energy, %  CP, %  Ca, %  P, %  0.60  0.55  6  76.5a,b  54.1  38.4a  33.9    0.75  6  75.9a,b  51.5  33.4b,c  34.8    0.95  6  77.6a  53.4  32.8b,c  37.3    1.15  6  76.5a,b  52.6  25.6d  26.4  0.45  0.55  6  78.0a  51.4  33.3b,c  37.2    0.75  6  76.6a,b  48.7  31.1c  35.6    0.95  6  76.3a,b  51.9  23.7d  34.8    1.15  6  74.8b  48.3  15.1e  31.9  Pooled SEM    0.57  1.42  1.30  1.67  TP, %  0.60  24  76.6  52.9a  32.5  33.1  0.45  24  76.4  50.0b  25.7  34.9  Pooled SEM    0.28  0.71  0.67  0.84  Ca, %  0.55  12  77.2  52.8  32.5  35.5a    0.75  12  76.2  50.1  30.3  35.2a    0.95  12  76.9  52.6  28.2  36.1a    1.15  12  75.6  50.5  20.3  29.1b  Pooled SEM    0.40  1.00  0.94  1.18  P-value  P    0.65  0.007  <0.0001  0.14  Ca    0.03  0.14  <0.0001  0.0005  Ca × P    0.02  0.81  0.01  0.11  TP level, %  Ca level, %  Reps  energy, %  CP, %  Ca, %  P, %  0.60  0.55  6  76.5a,b  54.1  38.4a  33.9    0.75  6  75.9a,b  51.5  33.4b,c  34.8    0.95  6  77.6a  53.4  32.8b,c  37.3    1.15  6  76.5a,b  52.6  25.6d  26.4  0.45  0.55  6  78.0a  51.4  33.3b,c  37.2    0.75  6  76.6a,b  48.7  31.1c  35.6    0.95  6  76.3a,b  51.9  23.7d  34.8    1.15  6  74.8b  48.3  15.1e  31.9  Pooled SEM    0.57  1.42  1.30  1.67  TP, %  0.60  24  76.6  52.9a  32.5  33.1  0.45  24  76.4  50.0b  25.7  34.9  Pooled SEM    0.28  0.71  0.67  0.84  Ca, %  0.55  12  77.2  52.8  32.5  35.5a    0.75  12  76.2  50.1  30.3  35.2a    0.95  12  76.9  52.6  28.2  36.1a    1.15  12  75.6  50.5  20.3  29.1b  Pooled SEM    0.40  1.00  0.94  1.18  P-value  P    0.65  0.007  <0.0001  0.14  Ca    0.03  0.14  <0.0001  0.0005  Ca × P    0.02  0.81  0.01  0.11  a–eMeans with different superscripts within the same column differ (P < 0.05). TP = total phosphorus; Ca = calcium; Reps = replicates; CP = crude protein. View Large DISCUSSION The depression of the growth performance and high mortality due to dietary P deficiency has been well demonstrated in the previous studies about P requirement in poultry (Waldroup et al., 2000; Yan et al., 2001; Liu, et al., 2017). In the present study, similar results were found in ducklings fed a low-P diet below the recommendation of NRC (1994) during the starter period. Some studies showed that high Ca levels had negative effects on growth performance in broilers that received diets with normal P levels (Rao et al., 2006; Hamdi et al., 2015), which was confirmed in ducklings in the current study. When under the low-P group, final BW, ADG, and ADFI were decreased as dietary Ca level increased from 0.55 to 1.15%. In addition, we found that high-Ca diet induced greater growth suppression in ducklings subjected to a low-P diet compared to the other 3 lower Ca diets. The magnitude of the depression may be due to the improper Ca to P ratios (Hamdi et al., 2015), because higher Ca to P ratios in diets tend to form the insoluble Ca-P complexes, resulting in reduced absorption of Ca and P (Simpson and Wise, 1990). However, the adverse effects of dietary high Ca to P ratios on growth performance were not observed in large domestic animals, such as cattle (Dowe et al., 1957) and gilts (Liptrap et al., 1970). It was suggested that poultry, with a shorter growth period, have greater sensitivity to dietary Ca excess. Therefore, the potential negative effect of higher Ca to P ratio in diets should be carefully considered when feeding diets with higher Ca level than the recommendation of NRC (1994), which were adopted for fast-growing strains to prevent skeletal problems and leg abnormalities in ducklings (Roberson et al., 2004). Faridi et al. (2015) has demonstrated that the negative effect of high Ca levels could be alleviated by increasing P content or improving P utilization (supplementation of phytase and vitamin D3) in diets. Serum Ca and P concentrations within a narrow physiological range are sensitive to reflect the body Ca and P metabolism (Veum, 2010). In the current study, the highest and lowest serum Ca concentrations were obtained in ducklings fed the diet with 0.95% Ca level under the normal- and low-P diets, respectively. The opposite results indicated that Ca and P ratio was a predominant factor in affecting Ca utilization. Under the normal-P diet, dietary Ca levels did not affect serum P concentration. However, under the low-P diet, serum P concentration was decreased as the dietary Ca increased from 0.55 to 0.95%, suggesting that increasing Ca level in the diet might inhibit P utilization and induced a greater P deficiency in ducks fed the low-P diet. The ALP enzyme activity, which is localized in the plasma membrane of osteoblasts before extracellular release, correlates with bone reabsorption (Golub and Boesze-Battaglia, 2007). The present study found that serum ALP activity was increased as dietary Ca level increased, indicating that ducklings might promote bone reabsorption to obtain P for important metabolism activities (energy metabolism, cellular signaling, membrane integrity, enzyme activity, etc.) when dietary P concentration is below the requirement. The highest serum ALP activity associated with the enhanced bone reabsorption might explain why serum P was increased at the high-Ca group compared to the medium-Ca group with a lower P diet. Calcitonin is a hormone known to participate in the regulation of Ca and P metabolism (Copp et al., 1962). In rats, P deprivation has been demonstrated to be a potent stimulus to serum CT secretion (Catherwood et al., 1983). In the current study, an elevated serum CT concentration was observed in ducklings fed a low-P diet. High CT concentration can inhibit osteoclast activity in bones (Yamamoto et al., 2005) and increase excretion of Ca and P in urinary (Carney, 1997), thus leading to the decreased serum Ca and P concentrations in the P-deficient ducklings. Bone mineralization was also influenced by dietary Ca and P in human and animals (Cumming, 1990; Hamdi et al., 2015). Similarly, in ducks, the low-P group decreased bone ash content and BMD compared to the normal-P group in our study. In broilers, the added Ca level from 0.40 to 0.90% in diets increased tibia bone ash content (Driver et al., 2005). However, high dietary Ca levels above the Ca requirement induced bone mineralization losses in broilers (Rao et al., 2006; Hamdi et al., 2015). In the present study, under the normal P diet, bone ash content was increased as dietary Ca content increased from 0.55 to 0.95%, but was decreased when at the highest Ca level with 1.15%. This effect might be explained by the reduction in Ca and P absorption associated with high Ca levels for bone mineralization (Liem et al., 2009). When ducklings from the low-P group were given a high-Ca diet, it further induced greater losses of bone ash content and BMD. These results agreed with those in the previous studies of broilers (Lei, et al., 1994; Sebastian et al., 1996). On the one hand, due to excess Ca in diets, the P utilization for bone mineralization was reduced by the formation of an insoluble complex in gut (Plumstead et al., 2008). On the other hand, more bone P was reabsorbed to maintain important metabolic actions when birds were fed high-Ca diets in P-deficient state (Cuisnier-Gleizes et al., 1976). In Exp. 2, we further evaluate the the utilization of energy, CP, Ca, and P of 8 diets used in Exp. 1 using the Sibbald method as described by Adeola et al. (1997). In the current study, low-P diet decreased the utilization of CP and Ca compared to normal-P diet, which may contribute to a poor feed conversion ratio observed in the ducks fed a low-P diet. In addition, a significant decrease in the utilization of Ca and P were observed in ducks fed high-Ca diet. As reported previously, a high dietary Ca level increased the intestinal pH and then decreased the apparent digestibility of Ca and P in broilers (Sebastian et al., 1996; Adeola and Walk, 2013) by the formation of insoluble Ca-P complexes (Plumstead et al., 2008). Under low P diet, the decline of Ca utilization at high-Ca diet became more aggravated, which might contribute to the greater loss of bone mineralization. In addition, under the low P diet, high-Ca diet decreased energy utilization of birds. 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Poultry ScienceOxford University Press

Published: Apr 1, 2018

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