Evaluation of dietary calcium level and source and phytase on growth performance, serum metabolites, and ileum mineral contents in broiler chicks fed adequate phosphorus diets from one to 28 days of age

Evaluation of dietary calcium level and source and phytase on growth performance, serum... Abstract A total of 936 one-day-old broiler chicks (Ross 308) was used to evaluate the effects of dietary calcium (Ca) source (1.0 and 0.6% from Ca carbonate [CC], or 0.6% from Celtic sea minerals [CSM]) on broiler chick response to phytase supplementation (0, 500, or 2,500 FTU per kg of diet) in phosphorus (P) adequate diets. Birds were randomly assigned to 9 treatments, each replicated 8 times (4 repeats of male and 4 repeats of female birds; 13 birds per replicate) in a completely randomized design. Results showed that birds fed low Ca CC diets had significantly (P ≤ 0.05) lower body weight at 14 and 21 d; weight gain during 1 to 14 d; feed intake during 1 to 14 d and 1 to 28 d; and toe and tibia ash content, ileum ash, and Ca, and P contents at 28 days. Feed conversion ratio and feed intake between 21 and 28 d, and serum Ca, Fe, and alkaline phosphatase levels at 28 d of age were not affected by dietary Ca level. The negative influence of reducing the dietary Ca level on body weight and weight gain was less severe when CC was replaced with CSM, and birds fed the CSM diet had a significantly lower feed conversion ratio and higher tibia P content compared to CC. Phytase did not have a significant influence (P > 0.05) on most measured parameters, but significantly reduced ileum P and ash contents, especially at the higher level of 2,500 vs. 500 units of phytase per kg of diet. These results confirm that CSM has a valuable potential to improve feed conversion ratio, and that phytase was less effective in compensating for a significantly reduced dietary Ca level as it is for P. INTRODUCTION It has been well documented that it is not only important to meet the dietary calcium (Ca) and phosphorus (P) requirements in broiler diet formulation, but also to target the appropriate ratio of Ca: P in order to ensure maximum performance and bone mineralization (Plumstead et al., 2008; Delezie et al., 2012; Dos Santos et al., 2013). Although the broiler chick needs more Ca than P, the higher cost of meeting its P demands and environmental concern relating to excess excreta P has motivated much more work on P than Ca. Such work has focused on the evaluation of different organic and inorganic P sources in order to determine their availability and at the same time the P requirement of the broiler. Work also has considered methods to improve the broilers’ ability to utilize plant derived P source via exogenous phytase (Selle and Ravindran, 2007; Plumstead et al., 2008; Delezie et al., 2012). However, the broiler chicks’ response to phytase is not straightforward, as it is influenced by many factors (Sebastian et al., 1998; Selle and Ravindran, 2007). Dietary Ca level is one of the most relevant feed items, as it not only markedly influences the broiler chicks’ P requirements, but also affects phytase efficacy (Dos Santos et al., 2013). Although some work has focused on Ca requirements, less work has been done to investigate the impact of dietary Ca source on phytase efficacy or explore the potential of phytase to enhance dietary Ca retention in low Ca diets (Augspurger and Baker, 2004; Dos Santos et al., 2013). Celtic sea mineral (CSM) is a calcareous marine algae (Lithothamnion calcareum), highly calcified with a mix of calcite, aragonite, and vaterite (Schlegel and Gutzwiller, 2016) and is an alternative inorganic source of Ca (up to 32% Ca). Though the Ca is the major mineral component of CSM, it also contains other minerals, such as Mg, Fe, P, Mn, I, Zn, etc. (González-Vega et al., 2014). Although many studies addressed the nutritional value of Ca carbonate, few studies reported the potential of CSM and its interaction with exogenous phytase in broiler chicks’ nutrition (González-Vega et al., 2014; Schlegel and Gutzwiller, 2016). Therefore, the aim of the present study was to determine the effects of different dietary Ca sources (Ca carbonate [CC] or CSM) on phytase efficacy in broiler chicks fed a P adequate diet. MATERIAL AND METHODS Diet Preparation Dietary treatments included a corn-soybean-meal-based diet (Table 1) with the recommended level of dietary Ca (1.0% Ca from CC; 0.6% from CC, and 0.6% of CSM source) in non-phytate P (nPP) adequate diets (0.50%). Each diet was supplemented with a graded level of phytase (0, 500, and 2,500 FTU per kg of the diet; Quantum phytase 5000 XT, AB-Vista Feed Ingredients, Marlborough, Wiltshire SN8 4AN, United Kingdom). The phytase supplement was a preparation of an evolved Escherichia coli 6-phytase (EC 3.1.2.26; 5,000 FTU g−1), produced by the genetically modified yeast Pichia pastoris. One unit of phytase activity was defined as the amount of enzyme that liberates one micromole of inorganic phosphate from sodium phytate at pH 5.5 and 37°C (Table 2). All enzymes were added to the representative basal diets as granules. To achieve maximum mixing uniformity, the enzyme was first mixed with a small quantity of each complete diet prior to incorporation into the mix with the rest of the diet. The CSM Ca source was obtained from Celtic Sea Minerals, Currabinny, Co Cork, Ireland, and is derived from Lithothmnion calcareum, a calcified sea weed derived product. Chicks and Rearing In this experiment, 936 one-day-old broiler chicks (Ross 308) were obtained from a local hatchery and randomly distributed among 9 treatments, each replicated 8 times (4 male replicates and 4 female replicates; each replicate contained 13 birds). The experiment lasted 28 days. All chicks were housed in 72 floor pens (1.25 × 1.4 m), with wood shavings used as litter. Birds received continuous lighting during the first 24 h, then maintained on a 23 L: 1D schedule during the rest of the experimental period. The experimental house temperature was maintained between 30 and 32°C at the beginning of the experiment and then gradually decreased by 2 to 3°C each wk to reach a final temperature of 22°C at the end. Chicks had free access to the mash diet and fresh water during the experiment. Care and management of the chicks were in accordance with commercial guidelines and were approved by University of Kurdistan Animal Ethics Committee. Diets Composition As described in Table 1, basal corn-soybean meal diets were formulated to meet or exceed the nutritional requirements of broiler chicks (NRC, 1994) for all nutrients with the exception of Ca. The experimental diets were formulated to contain different Ca levels derived from 2 different Ca sources, CC and CSM. Dietary Ca levels investigated were 1% from CC, 0.6% from CC, and 0.6% from CSM. Basal diets were formulated to be isonitrogenous, isocaloric, and isophosphoric (0.50% nPP). The nutritional composition of experimental diets are shown in Table 1. Table 1. Composition (%) and calculated nutrient content of experimental diets.   High calcium  Low calcium    Calcium  Calcium  Celtic sea    carbonate  carbonate  mineral  Ingredient  (CC)  (CC)  (CSM)  Corn  58.95  61.04  60.57  Soybean meal 46  33.18  32.83  32.91  Soy oil  2.79  2.09  2.25  Salt  0.28  0.28  0.28  DL Methionine  0.32  0.32  0.32  Lysine HCl  0.27  0.28  0.27  Threonine  0.07  0.07  0.07  Calcium carbonate1  1.97  0.92  0.00  Dicalcium Phosphate  0.65  0.65  0.65  Celtic sea mineral2  0.00  0.00  1.16  Phosphoric acid  1.00  1.00  1.00  Coccidiostat (Coban - monensin)  0.02  0.02  0.02  Vitamin premix3  0.25  0.25  0.25  Mineral premix4  0.25  0.25  0.25  Calculated analysis  Crude protein %  21.40  21.40  21.40  Poult ME kcal/kg  3085  3085  3085  Calcium %  1.00  0.60  0.60  Phos %  0.79  0.79  0.79  Avail phos %  0.50  0.50  0.50  Arg %  1.41  1.41  1.41  Cysteine, %  0.35  0.35  0.35  Lysine, %  1.35  1.35  1.35  Methionine, %  0.65  0.65  0.65  Total sulphur amino acids, %  1.00  1.00  1.00  Tryp %  0.24  0.24  0.24  Na %  0.18  0.18  0.18  Cl %  0.26  0.26  0.26  K %  0.91  0.90  0.90  Analyzed nutrients, %  Dry matter, %  89.66  89.54  89.52  Ash, %  5.68  5.35  5.26  Crude protein, %  19.67  19.66  20.05  Ca, %  1.15  0.77  0.75  Total P,%  0.73  0.70  0.74    High calcium  Low calcium    Calcium  Calcium  Celtic sea    carbonate  carbonate  mineral  Ingredient  (CC)  (CC)  (CSM)  Corn  58.95  61.04  60.57  Soybean meal 46  33.18  32.83  32.91  Soy oil  2.79  2.09  2.25  Salt  0.28  0.28  0.28  DL Methionine  0.32  0.32  0.32  Lysine HCl  0.27  0.28  0.27  Threonine  0.07  0.07  0.07  Calcium carbonate1  1.97  0.92  0.00  Dicalcium Phosphate  0.65  0.65  0.65  Celtic sea mineral2  0.00  0.00  1.16  Phosphoric acid  1.00  1.00  1.00  Coccidiostat (Coban - monensin)  0.02  0.02  0.02  Vitamin premix3  0.25  0.25  0.25  Mineral premix4  0.25  0.25  0.25  Calculated analysis  Crude protein %  21.40  21.40  21.40  Poult ME kcal/kg  3085  3085  3085  Calcium %  1.00  0.60  0.60  Phos %  0.79  0.79  0.79  Avail phos %  0.50  0.50  0.50  Arg %  1.41  1.41  1.41  Cysteine, %  0.35  0.35  0.35  Lysine, %  1.35  1.35  1.35  Methionine, %  0.65  0.65  0.65  Total sulphur amino acids, %  1.00  1.00  1.00  Tryp %  0.24  0.24  0.24  Na %  0.18  0.18  0.18  Cl %  0.26  0.26  0.26  K %  0.91  0.90  0.90  Analyzed nutrients, %  Dry matter, %  89.66  89.54  89.52  Ash, %  5.68  5.35  5.26  Crude protein, %  19.67  19.66  20.05  Ca, %  1.15  0.77  0.75  Total P,%  0.73  0.70  0.74  1Calcium carbonate, 38% Ca. 2Celtic sea mineral, 32.5% Ca. 3Provides per kg of diet: Vit. A (as all-trans retinol acetate), 9,000 I.U.; Cholecalciferol, 2,000 I.U.; Vit. E (as dl- alpha-tocopheryl acetate), 18 I.U.; Vit K (as menadion sodium bisulfate), 2 mg; Thiamine (as thiamin mononitrate), 1.8 mg; Riboflavin, 6.6 mg; Niacin, 30 mg; Pyridoxin, 3 mg; Vit B12, 15 mcg; Calcium d-Pantothenate, 10 mg; Folic acid, 1 mg; Biotin (as d-biotin), 0.1 mg; Choline chloride (as choline chloride), 500 mg; Antioxidant (as butylatedhydroxy toluene), 100 mg. 4Provides per Kg of diet: Manganese (as MnO), 100 mg; Zinc (ZnSO4. 7H2O), 84.7 mg; Iron (FeSO4. 7H2O), 50 mg; Copper (CuSO4. 5H2O), 10 mg; Iodine (KI), 1 mg; Se (Na2SeO3), 0.2 mg. View Large Table 2. Phytase activity in experimental diets.1 Dietary treatments  Phytase recovery  Ca Source  Ca Level  Phytase  (FTU. Kg−1)    (%)  (U.Kg−1)    CC  1.0  0  <50  CC  1.0  500  526  CC  1.0  2,500  2,263  CC  0.6  0  ∼93  CC  0.6  500  711  CC  0.6  2,500  2,257  CSM  0.6  0  ∼72  CSM  0.6  500  543  CSM  0.6  2,500  2,548  Dietary treatments  Phytase recovery  Ca Source  Ca Level  Phytase  (FTU. Kg−1)    (%)  (U.Kg−1)    CC  1.0  0  <50  CC  1.0  500  526  CC  1.0  2,500  2,263  CC  0.6  0  ∼93  CC  0.6  500  711  CC  0.6  2,500  2,257  CSM  0.6  0  ∼72  CSM  0.6  500  543  CSM  0.6  2,500  2,548  1Dietary phytase quantification determined using ELISA method by AB Vista on final diets (Engelen et al., 2001). CSM: Celtic sea minerals, CC: Calcium carbonate. View Large Measurements Birds were weighed as a group on arrival and at 14, 21, and 28 d of age on a pen basis. Feed intake was also recorded at the same time points for calculation of feed conversion ratio after adjustment for the weight of dead birds in each growth period. Average body weight, body weight gain, feed intake, and feed conversion ratio were determined between 1 to 14, 14 to 21, 21 to 28, and 1 to 28 d of age. At 28 d of age, 3 birds per pen were randomly selected for blood sampling from the left wing vein. After sampling, these selected birds were killed by cervical dislocation, and the right and left tibia and all toe bones were removed. Subsequently, tibias were de-fleshed and defatted in a mixture of 90% ethyl ether and 10% methanol. Finally, pooled toe and defatted tibia bones were dried at 105°C until a consistent weight was obtained, and then ashed in a muffled furnace at 605°C for at least 12 h (AOAC, 1990). The ash content was expressed as g of ash per 100 g of the dried weight for each bone. At the same time, ileum digesta samples from all sacrificed birds were collected for ash, Ca, and P analysis. The Ca and P contents of dried tibia and ileum digesta were measured (AOAC, 1990). Blood serum P, Ca, Fe, and alkaline phosphatase were measured using enzymatic, colorimetric essays using the relevant clinical kits (Pars Azmun, Tehran, Iran). The chemical composition of experimental diets, including DM, CP, Ca, P (AOAC, 1990), and phytase (Engelen et al., 2001), also were measured as described in Table 1. Statistical Analysis Data were analyzed using the General Linear Models (GLM) procedure of SAS (SAS institute, 1991) using a completely randomized design (CRD) in a factorial arrangement. Mortality data were transformed using $$\sqrt {X + 1}$$ prior to analysis (Manikandan, 2010). Significant differences among treatments were determined at P ≤ 0.05 using Tukey tests. Additional analyses were conducted using contrast statements to examine the effects of Ca source at 0.6% Ca inclusion (CC vs. CSM) or Ca level (1.0 vs. 0.6%). The dose-response effect of supplemental phytase was computed using orthogonal polynomial contrast for liner and quadratic effects (SAS, 1991). RESULTS Performance No significant interaction effects of treatment on performance were noted when the whole data set was considered, but in contrast, comparing Ca levels between the 2 CC diets, it was clear that the lower level (0.6%) led to significantly lower weight gain (1 to 14, 14 to 21, and 1 to 28 d) and body weights (14, 21, and 28 d) compared with the 1% control. No differences between calcium sources or phytase levels were found (P > 0.05) on body weight or gain (Table 3). Table 3. Effects of calcium source, level, and phytase supplementation on body weight and weight gain (g) in broiler chicks fed high phosphorus diets from 1 to 28 d of age.     Body weight (g)  Weight gain (g)      1 d  14 d  21 d  28 d  1 to 14 d  14 to 21 d  21 to 28 d  1 to 28 d  Ca Source   CC, 1.0% Ca    44  407a  828a  1313  363a  421  485  1269   CC, 0.60% Ca    45  389b  787b  1245  345b  398  458  1201   CSM, 0.60% Ca    44  400a,b  810a,b  1288  355a,b  411  478  1244  Phytase level (U. Kg−1)   0.0    44  394  804  1278  350  410  475  1234   500    44  404  816  1293  360  412  477  1248   2,500    44  398  806  1275  354  409  469  1231  Ca source × Phytase level   CC, 1.0% Ca  0  45  412  843  1325  368  430  483  1281    500  44  414  831  1327  370  417  496  1283    2,500  44  396  811  1287  352  415  476  1243   CC, 0.6% Ca  0  44  381  776  1232  337  395  457  1188    500  44  401  817  1289  357  416  472  1245    2,500  44  384  768  1213  340  384  445  1169   CSM, 0.6% Ca  0  44  389  793  1278  345  404  485  1233    500  44  397  798  1261  353  401  463  1217    2,500  44  413  840  1326  369  427  486  1281   SEM    0.22  9.08  20.23  36.67  9.03  12.70  19.84  36.62   Ca source (Ca)    0.574  0.046  0.049  0.077  0.047  0.099  0.231  0.077   Phytase level (P)    0.307  0.401  0.750  0.828  0.380  0.961  0.879  0.825   Ca × P    0.605  0.150  0.136  0.369  0.144  0.176  0.725  0.366  Source of variation of phytase level, P-value   Linear    0.150  0.183  0.471  0.634  0.170  0.847  0.886  0.628   Quadratic    0.593  0.832  0.823  0.700  0.840  0.838  0.628  0.702  Orthogonal contrasts   CC, 1.0% vs. CC, 0.6%    0.406  0.014  0.015  0.026  0.014  0.033  0.100  0.026   CC, 0.6% vs. CSM, 0.6%    0.331  0.144  0.163  0.153  0.149  0.238  0.222  0.154   CC, 1.0% vs. CSM, 0.6%    0.888  0.300  0.277  0.404  0.293  0.324  0.665  0.403      Body weight (g)  Weight gain (g)      1 d  14 d  21 d  28 d  1 to 14 d  14 to 21 d  21 to 28 d  1 to 28 d  Ca Source   CC, 1.0% Ca    44  407a  828a  1313  363a  421  485  1269   CC, 0.60% Ca    45  389b  787b  1245  345b  398  458  1201   CSM, 0.60% Ca    44  400a,b  810a,b  1288  355a,b  411  478  1244  Phytase level (U. Kg−1)   0.0    44  394  804  1278  350  410  475  1234   500    44  404  816  1293  360  412  477  1248   2,500    44  398  806  1275  354  409  469  1231  Ca source × Phytase level   CC, 1.0% Ca  0  45  412  843  1325  368  430  483  1281    500  44  414  831  1327  370  417  496  1283    2,500  44  396  811  1287  352  415  476  1243   CC, 0.6% Ca  0  44  381  776  1232  337  395  457  1188    500  44  401  817  1289  357  416  472  1245    2,500  44  384  768  1213  340  384  445  1169   CSM, 0.6% Ca  0  44  389  793  1278  345  404  485  1233    500  44  397  798  1261  353  401  463  1217    2,500  44  413  840  1326  369  427  486  1281   SEM    0.22  9.08  20.23  36.67  9.03  12.70  19.84  36.62   Ca source (Ca)    0.574  0.046  0.049  0.077  0.047  0.099  0.231  0.077   Phytase level (P)    0.307  0.401  0.750  0.828  0.380  0.961  0.879  0.825   Ca × P    0.605  0.150  0.136  0.369  0.144  0.176  0.725  0.366  Source of variation of phytase level, P-value   Linear    0.150  0.183  0.471  0.634  0.170  0.847  0.886  0.628   Quadratic    0.593  0.832  0.823  0.700  0.840  0.838  0.628  0.702  Orthogonal contrasts   CC, 1.0% vs. CC, 0.6%    0.406  0.014  0.015  0.026  0.014  0.033  0.100  0.026   CC, 0.6% vs. CSM, 0.6%    0.331  0.144  0.163  0.153  0.149  0.238  0.222  0.154   CC, 1.0% vs. CSM, 0.6%    0.888  0.300  0.277  0.404  0.293  0.324  0.665  0.403  a,bMeans in column and under each main effects with common superscript do not differ significantly (P ≤ 0.05). CSM: Celtic sea minerals, CC: Calcium carbonate. View Large Treatment effects on feed intake were noted between 1 to 14, 14 to 21, and 1 to 28 d of age only (P ≤ 0.05), Reducing dietary Ca level from 1.0 to 0.6% reduced (P ≤ 0.05) feed intake between 1 to 14 (538 vs. 572 g), 14 to 21 (657 vs. 690), and 1 to 28 (2,057 vs. 2,156) d but not during the last wk (21 to 28 d). Phytase did not influence feed intake during any period (P > 0.05). Treatment effects on feed conversion were detected between 14 to 21 and 1 to 28 d of age (P ≤ 0.05). The contrast data suggest that FCR was significantly better for birds fed the CSM diet compared with the same amount of Ca derived from the CC diet (P ≤ 0.05). Feed conversion ratio was not influenced by the addition of phytase. As presented in Table 3 and Table 4, addition of different levels of phytase had no linear or quadratic effects on performance criteria. Mortality (%) was not influenced by dietary Ca level (1.0 vs. 0.6%) or Ca source (data not shown). Table 4. Effects of calcium source level and phytase supplementation on feed intake (g) and feed conversion ratio (g. g−1) in broiler chicks fed a high phosphorus diet from 1 to 28 d of age.     Feed intake (g)  Feed conversion ratio (g. g−1)      1 to 14 d  14 to 21 d  21 to 28 d  1 to 28 d  1 to 14 d  14 to 21 d  21 to 28 d  1 to 28 d  Ca Source   CC, 1.0% Ca    572a  690a  905  2156a  1.58  1.64a,b  1.88  1.70a   CC, 0.60% Ca    538b  657a,b  873  2057b  1.56  1.66a  1.92  1.72a   CSM, 0.60% Ca    538b  651b  876  2052b  1.52  1.59b  1.84  1.65b  Phytase level (U. Kg−1)   0.0    546  669  877  2080  1.56  1.64  1.86  1.69   500    556  672  900  2115  1.54  1.64  1.90  1.70   2,500    546  658  877  2070  1.56  1.62  1.88  1.69  Ca source × Phytase level   CC, 1.0% Ca  0  568  699  910  2165  1.55  1.62  1.89  1.69    500  574  685  931  2177  1.55  1.65  1.88  1.70    2,500  573  686  873  2126  1.64  1.65  1.86  1.71   CC, 0.6% Ca  0  534  658  855  2037  1.58  1.67  1.88  1.72    500  563  681  913  2145  1.56  1.64  1.95  1.73    2,500  517  633  852  1989  1.54  1.66  1.93  1.71   CSM, 0.6% Ca  0  535  649  866  2038  1.56  1.61  1.80  1.66    500  531  649  856  2024  1.50  1.62  1.86  1.67    2,500  548  656  906  2095  1.50  1.54  1.87  1.64   SEM    11.93  17.48  26.15  46.91  0.03  0.03  0.05  0.02   Ca source (Ca)    0.001  0.019  0.270  0.013  0.115  0.030  0.166  0.004   Phytase level (P)    0.511  0.612  0.458  0.465  0.634  0.740  0.625  0.857   Ca × P    0.135  0.506  0.183  0.199  0.193  0.354  0.861  0.870  Source of variation of phytase level, P-value   Linear    0.305  0.818  0.278  0.360  0.374  0.966  0.344  0.679   Quadratic    0.593  0.337  0.537  0.405  0.732  0.440  0.850  0.714  Orthogonal contrasts   CC, 1.0% vs. CC, 0.6%    0.001  0.026  0.146  0.012  0.459  0.568  0.330  0.416   CC, 0.6% vs. CSM, 0.6%    0.978  0.682  0.902  0.909  0.184  0.012  0.059  0.001   CC, 1.0% vs. CSM, 0.6%    0.001  0.009  0.182  0.009  0.041  0.048  0.351  0.014      Feed intake (g)  Feed conversion ratio (g. g−1)      1 to 14 d  14 to 21 d  21 to 28 d  1 to 28 d  1 to 14 d  14 to 21 d  21 to 28 d  1 to 28 d  Ca Source   CC, 1.0% Ca    572a  690a  905  2156a  1.58  1.64a,b  1.88  1.70a   CC, 0.60% Ca    538b  657a,b  873  2057b  1.56  1.66a  1.92  1.72a   CSM, 0.60% Ca    538b  651b  876  2052b  1.52  1.59b  1.84  1.65b  Phytase level (U. Kg−1)   0.0    546  669  877  2080  1.56  1.64  1.86  1.69   500    556  672  900  2115  1.54  1.64  1.90  1.70   2,500    546  658  877  2070  1.56  1.62  1.88  1.69  Ca source × Phytase level   CC, 1.0% Ca  0  568  699  910  2165  1.55  1.62  1.89  1.69    500  574  685  931  2177  1.55  1.65  1.88  1.70    2,500  573  686  873  2126  1.64  1.65  1.86  1.71   CC, 0.6% Ca  0  534  658  855  2037  1.58  1.67  1.88  1.72    500  563  681  913  2145  1.56  1.64  1.95  1.73    2,500  517  633  852  1989  1.54  1.66  1.93  1.71   CSM, 0.6% Ca  0  535  649  866  2038  1.56  1.61  1.80  1.66    500  531  649  856  2024  1.50  1.62  1.86  1.67    2,500  548  656  906  2095  1.50  1.54  1.87  1.64   SEM    11.93  17.48  26.15  46.91  0.03  0.03  0.05  0.02   Ca source (Ca)    0.001  0.019  0.270  0.013  0.115  0.030  0.166  0.004   Phytase level (P)    0.511  0.612  0.458  0.465  0.634  0.740  0.625  0.857   Ca × P    0.135  0.506  0.183  0.199  0.193  0.354  0.861  0.870  Source of variation of phytase level, P-value   Linear    0.305  0.818  0.278  0.360  0.374  0.966  0.344  0.679   Quadratic    0.593  0.337  0.537  0.405  0.732  0.440  0.850  0.714  Orthogonal contrasts   CC, 1.0% vs. CC, 0.6%    0.001  0.026  0.146  0.012  0.459  0.568  0.330  0.416   CC, 0.6% vs. CSM, 0.6%    0.978  0.682  0.902  0.909  0.184  0.012  0.059  0.001   CC, 1.0% vs. CSM, 0.6%    0.001  0.009  0.182  0.009  0.041  0.048  0.351  0.014  a,bMeans in column and under each main effects with common superscript do not differ significantly (P ≤ 0.05). CSM: Celtic sea minerals, CC: Calcium carbonate. View Large Bone Mineralization No significant treatment effects were noted for tibia ash, although the results suggested there was a significant reduction in toe ash when (P ≤ 0.05) the dietary Ca level was reduced from 1.0 to 0.6%. Phytase supplementation had no significant (P > 0.05) effect on toe ash content (P > 0.05). As shown in Table 5, though tibia ash content (%) at 28 d of age was not influenced by treatment, the contrast of the low vs. high CC Ca level suggested the latter had greater ash content (P ≤ 0.05). Phytase had no effect, and there were no differences (P > 0.05) between the 2 Ca sources noted for tibia ash (Table 5). Table 5. Effects of calcium source level and phytase supplementation on broiler chicks bone mineralization and ileum mineral contents at 28 d of age.     Toe Ash (%)  Tibia contents (%)  Ileum content (%)        Ash  Ca  P  Ash  Ca  P  Ca Source   CC, 1.0% Ca    14.3a  37.8  10.3b  6.1a,b  11.7a  0.47a  1.14a   CC, 0.60% Ca    13.2b  36.7  11.3a  5.9b  8.9b  0.27b  0.88b   CSM, 0.60% Ca    13.3b  37.5  11.4a  6.2a  8.9b  0.29b  0.85b  Phytase level (U. Kg−1)   0.0    13.8  37.5  11.5  6.1  10.4a  0.34  1.07a   500    14.0  37.1  10.3  6.0  10.0a  0.35  0.96b   2,500    13.1  37.4  11.2  6.1  9.2b  0.34  0.85c  Ca source × Phytase level   CC, 1.0% Ca  0  15.4  38.1  10.7a,b  6.1  12.0  0.51  1.18a,b    500  14.1  37.5  9.0b  6.1  12.4  0.47  1.21a    2,500  13.5  37.9  11.1a,b  6.0  10.7  0.44  1.04a–c   CC, 0.6% Ca  0  13.1  37.4  12.7a  6.0  9.2  0.25  0.98b–d    500  13.9  36.3  10.9a,b  5.9  9.1  0.28  0.87d,e    2,500  12.8  36.4  10.3a,b  5.7  8.1  0.26  0.79d,e   CSM, 0.6% Ca  0  13.1  36.9  11.1a,b  6.1  9.4  0.27  1.03a–c    500  13.9  37.7  11.0a,b  6.0  8.6  0.29  0.79d,e    2,500  13.0  37.8  12.1a  6.5  8.7  0.31  0.73e   SEM                   Ca source (Ca)    0.046  0.099  0.045  0.013  <0.0001  <0.0001  <0.0001   Phytase level (P)    0.156  0.820  0.059  0.773  0.001  0.920  <0.0001   Ca × P    0.323  0.556  0.028  0.065  0.218  0.424  <0.048  Source of variation of phytase level, P-value   Linear    0.777  0.544  0.024  0.582  0.338  0.817  <0.006   Quadratic    0.057  0.870  0.477  0.653  0.0003  0.740  <0.0001  Orthogonal contrasts   CC, 1.0% vs. CC, 0.6%    0.023  0.037  0.042  0.047  <0.0001  <0.0001  <0.0001   CC, 0.6% vs. CSM, 0.6%    0.855  0.136  0.782  0.004  0.995  0.275  0.497   CC, 1.0% vs. CSM, 0.6%    0.043  0.540  0.024  0.355  <0.0001  <0.0001  <0.0001      Toe Ash (%)  Tibia contents (%)  Ileum content (%)        Ash  Ca  P  Ash  Ca  P  Ca Source   CC, 1.0% Ca    14.3a  37.8  10.3b  6.1a,b  11.7a  0.47a  1.14a   CC, 0.60% Ca    13.2b  36.7  11.3a  5.9b  8.9b  0.27b  0.88b   CSM, 0.60% Ca    13.3b  37.5  11.4a  6.2a  8.9b  0.29b  0.85b  Phytase level (U. Kg−1)   0.0    13.8  37.5  11.5  6.1  10.4a  0.34  1.07a   500    14.0  37.1  10.3  6.0  10.0a  0.35  0.96b   2,500    13.1  37.4  11.2  6.1  9.2b  0.34  0.85c  Ca source × Phytase level   CC, 1.0% Ca  0  15.4  38.1  10.7a,b  6.1  12.0  0.51  1.18a,b    500  14.1  37.5  9.0b  6.1  12.4  0.47  1.21a    2,500  13.5  37.9  11.1a,b  6.0  10.7  0.44  1.04a–c   CC, 0.6% Ca  0  13.1  37.4  12.7a  6.0  9.2  0.25  0.98b–d    500  13.9  36.3  10.9a,b  5.9  9.1  0.28  0.87d,e    2,500  12.8  36.4  10.3a,b  5.7  8.1  0.26  0.79d,e   CSM, 0.6% Ca  0  13.1  36.9  11.1a,b  6.1  9.4  0.27  1.03a–c    500  13.9  37.7  11.0a,b  6.0  8.6  0.29  0.79d,e    2,500  13.0  37.8  12.1a  6.5  8.7  0.31  0.73e   SEM                   Ca source (Ca)    0.046  0.099  0.045  0.013  <0.0001  <0.0001  <0.0001   Phytase level (P)    0.156  0.820  0.059  0.773  0.001  0.920  <0.0001   Ca × P    0.323  0.556  0.028  0.065  0.218  0.424  <0.048  Source of variation of phytase level, P-value   Linear    0.777  0.544  0.024  0.582  0.338  0.817  <0.006   Quadratic    0.057  0.870  0.477  0.653  0.0003  0.740  <0.0001  Orthogonal contrasts   CC, 1.0% vs. CC, 0.6%    0.023  0.037  0.042  0.047  <0.0001  <0.0001  <0.0001   CC, 0.6% vs. CSM, 0.6%    0.855  0.136  0.782  0.004  0.995  0.275  0.497   CC, 1.0% vs. CSM, 0.6%    0.043  0.540  0.024  0.355  <0.0001  <0.0001  <0.0001  a–eMeans in column and under each main effects with common superscript do not differ significantly (P ≤ 0.05). CSM: Celtic sea minerals, CC: Calcium carbonate. View Large Tibia Ca content (%) was significantly influenced by dietary Ca source × phytase level interaction (P < 0.05), which seemed to be due to lower levels being found in the 500 FTU CC 1% diet compared with the unsupplemented CC 0.6% diet and the 2,500 FTU supplemented CSM diet. Tibia P was minimized in the lower Ca CC diets. As presented in Tables 3 and 4, supplementation of different levels of phytase to the basal diet had no linear or quadratic effect. Mineral Content of the Ileum Ileal content of ash, Ca, and P was significantly influenced by reduction of dietary Ca level, regardless of Ca source. In addition, phytase supplementation reduced ash and P contents of the ileum, particularly at the highest dose in the CSM diet. As noted in Table 5, addition of graded levels of phytase to the basal diet resulted in linear and quadratic reduction in ileum P content. Serum Metabolites As shown in Table 6, there were no significant treatment effects on serum traits at 28 d of age, although the effect of Ca level reduction on serum P was significant. Serum P was lower in birds fed the high Ca CC diets compared with the low Ca CC diets. No effects of phytase on serum traits were noted (P > 0.05). Table 6. Effects of calcium source level and phytase supplementation on blood serum components of broiler chicks at 28 d of age.     Calcium (mg/ dl)  Fe (μg/dl)  Alkaline phosphatase (IU/l)  Phosphorus (mg/dl)  Ca Source   CC, 1.0% Ca    8.8  89.2  2906  5.9b   CC, 0.60% Ca    8.7  87.4  3129  6.5a,b   CSM, 0.60% Ca    9.0  95.1  2860  6.6a  Phytase level (U. Kg−1)   0.0    9.1  88.4  2660  6.2   500    8.9  95.4  3062  6.2   2,500    8.4  88.1  3217  6.5  Ca source × Phytase level   CC, 1.0% Ca  0  9.2  83.3  2654.8  6.1    500  9.1  103.8  2688.0  5.9    2,500  8.1  77.3  3338.2  5.8   CC, 0.6% Ca  0  9.1  81.8  2845.1  6.1    500  8.9  95.9  3576.7  6.5    2,500  8.2  86.4  3076.3  6.8   CSM, 0.6% Ca  0  9.2  101.3  2455.6  6.4    500  8.7  87.7  2920.5  6.3    2,500  9.1  97.3  3213.6  7.1   SEM      0.41  10.05  451.16   Ca source (Ca)    0.715  0.572  0.672  0.019   Phytase level (P)    0.127  0.528  0.260  0.271   Ca × P    0.459  0.317  0.763  0.248  Source of variation of phytase level, P-value   Linear    0.506  0.376  0.277  0.944   Quadratic    0.055  0.477  0.249  0.108  Orthogonal contrasts   CC, 1.0% vs. CC, 0.6%    0.896  0.998  0.482  0.049   CC, 0.6% vs. CSM, 0.6%    0.446  0.363  0.415  0.435   CC, 1.0% vs. CSM, 0.6%    0.521  0.371  0.905  0.007      Calcium (mg/ dl)  Fe (μg/dl)  Alkaline phosphatase (IU/l)  Phosphorus (mg/dl)  Ca Source   CC, 1.0% Ca    8.8  89.2  2906  5.9b   CC, 0.60% Ca    8.7  87.4  3129  6.5a,b   CSM, 0.60% Ca    9.0  95.1  2860  6.6a  Phytase level (U. Kg−1)   0.0    9.1  88.4  2660  6.2   500    8.9  95.4  3062  6.2   2,500    8.4  88.1  3217  6.5  Ca source × Phytase level   CC, 1.0% Ca  0  9.2  83.3  2654.8  6.1    500  9.1  103.8  2688.0  5.9    2,500  8.1  77.3  3338.2  5.8   CC, 0.6% Ca  0  9.1  81.8  2845.1  6.1    500  8.9  95.9  3576.7  6.5    2,500  8.2  86.4  3076.3  6.8   CSM, 0.6% Ca  0  9.2  101.3  2455.6  6.4    500  8.7  87.7  2920.5  6.3    2,500  9.1  97.3  3213.6  7.1   SEM      0.41  10.05  451.16   Ca source (Ca)    0.715  0.572  0.672  0.019   Phytase level (P)    0.127  0.528  0.260  0.271   Ca × P    0.459  0.317  0.763  0.248  Source of variation of phytase level, P-value   Linear    0.506  0.376  0.277  0.944   Quadratic    0.055  0.477  0.249  0.108  Orthogonal contrasts   CC, 1.0% vs. CC, 0.6%    0.896  0.998  0.482  0.049   CC, 0.6% vs. CSM, 0.6%    0.446  0.363  0.415  0.435   CC, 1.0% vs. CSM, 0.6%    0.521  0.371  0.905  0.007  a,bMeans in column and under each main effects with common superscript do not differ significantly (P ≤ 0.05). CSM: Celtic sea minerals, CC: Calcium carbonate. View Large DISCUSSION The findings of the current experiment with regard to the negative consequences of reducing dietary Ca from 1.0 to 0.6% in a P adequate diet (0.5%) on body weight, weight gain, feed intake, toe and tibia ash contents are in agreement with previously published reports (Driver et al., 2005; Delezie et al., 2012). These data indicate that the Ca deficiency in the current experiment when CC was fed at 0.6% was moderate and likely a result of the fact that the P levels were simultaneously luxurious. The analyzed Ca content of the low Ca diets was approximately 0.77%, which would be deemed adequate if the available phosphate levels were approximately 0.35%, but in this study they were formulated to be much higher at 0.5%. In addition, the data confirm that some criteria, such as body weight, are more responsive to dietary Ca level than feed conversion ratio and serum mineral contents (Dos Santos et al., 2013). Driver et al. (2005) demonstrated that reducing dietary Ca level to less than 0.625% in a high P diet (0.45%) had negative effects on body weight to 16 d of age, but in lower P diets, this detrimental effect was reduced. Augspurger and Baker (2004) also showed that reducing dietary Ca level from 1 to 0.48% in a high nPP diet (0.45%) significantly reduced weight gain. It has been postulated that the negative impacts of reducing dietary Ca level on broiler performance are just as much related to disturbing the dietary Ca: available P balance as it is to shortage of Ca supply. Thus, the effects of feeding a particular dietary Ca level needs to take into account the P level of the diet, as this dictates whether an appropriate Ca: available P ratio has been achieved (Shafey, 1993; Augspurger and Baker, 2004). Some researchers suggest that the absolute Ca and P requirements of broiler chicks is less than the current NRC (1994) recommendation or broiler company nutrient specifications. As a result, the observed adverse effects noted in some research of feeding low Ca diets to young broiler chicks is more of a consequence of a narrow dietary Ca: P balance rather than a Ca deficiency per se (Delezie et al., 2012). Delezie et al. (2012) reported that concomitant and coordinated reduction of dietary Ca and P levels had no deleterious effects on bone mineralization, but feeding an imbalanced Ca and P diet significantly reduced body weight, bone development, and Ca and P retention. The current findings that CSM was better than CC as a Ca source in a low Ca diet, with lower feed conversion ratios, is partially explained by higher solubility of CSM compared with CC (Walk et al., 2012). This would contribute to correcting the Ca: P ratio imbalance caused by feeding a low Ca, high P level in the basal diet. Use of a dietary Ca source such as CSM, which has higher Ca solubility than commonly used Ca sources such as CC, will enable the use of lower Ca diets without imbalancing the dietary Ca: P ratio. There were few if any beneficial effects of feeding phytase on performance or tibia ash content (%) in this study. Use of this phytase at 500 FTU would be expected to release Ca and P in an approximate 1:1 ratio, which, in the low Ca diets, would further imbalance the Ca: P ratio. Even though the amount of digestible Ca would be increased, this benefit is overshadowed by the negative effect of the incremental P digestibility on the ratio between Ca and P. Indeed, phytase supplementation, especially at the highest level, significantly reduced ileum ash and P content, suggesting better absorption, but it was less effective on ileum Ca status. The lack of effect of phytase on performance indicates that the birds did not suffer a deficiency of P. Under the circumstances of this trial, the P released by the phytase in the ileum was likely absorbed, but much of it would likely be excreted in the urine if there was not sufficient Ca with which to combine for bone deposition. Thus, there may have been an increase in the soluble P in excreta of such birds (Selle and Ravindran, 2007). Phytate, a myo-inositol ring with 6 phosphate groups attached, has enormous capacity to bind cations (such as Ca, P, Cu, and Zn) and form insoluble mineral-phytate complexes along with complexing with other nutrients, such as starch and amino acids, rendering them less digestible (Sebastian et al., 1998; Selle and Ravindran, 2007). The ability of poultry to hydrolyze the aforementioned complexes and release the bound nutrients depends on bird age, feed ingredients used, level of other nutrients, such as Ca and P, and also level of exogenous phytase supplementation (Rousseau et al., 2012; Liu et al., 2013; Walk et al., 2014). Exogenous phytase efficacy also depends upon its application dosage and dietary Ca level. In fact, keeping dietary Ca levels high, while maintaining dietary non-phytate P at a minimum level is the most effective way of compromising performance to such an extent that the benefits of added microbial phytase is maximized (Applegate et al., 2003; Plumstead et al., 2008). It has been postulated that reducing dietary Ca level significantly reduces the possibility of formation of insoluble-phytate complexes and thus more phytate is hydrolyzed in the intestinal tract by endogenous phytase (Applegate et al. 2003). This reduces the scope for an exogenous phytase response. On the other hand, phytase efficacy is highly dependent on dietary nPP level, with little effect noted in high P diets as was the case in the current trial. Therefore, since the basal diet had sufficient P to support the birds’ requirements, the lack of a significant effect of phytase in the present experiment is expected. However, the effects of phytase on reducing ileum ash and P contents indicates the functionality of the phytase even though a significant proportion of the absorbed P was probably excreted. In conclusion, our findings indicates that using a dietary Ca source such as CSM instead of CC allows use of a lower Ca diet with few consequences. When phytase is used in such diets, even at very high dosages, few benefits were observed, likely as a result of an imbalance between Ca and P, which was possibly made worse by phytase addition. Acknowledgements We gratefully acknowledge AB-Vista Feed Ingredients, (Marlborough, Wiltshire, SN8 4AN, United Kingdom) for providing phytase and CSM samples. REFERENCES AOAC. 1990. Official Methods of Analysis . 15th ed. Association of Official Analytical Chemists, Arlington, Virginia, USA. Applegate T. J., Angel R., Classen H. L.. 2003. 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L., Addo-Chidie E. K., Bedford M. R., Adeola O.. 2012. Evaluation of a highly soluble calcium source and phytase in the diets of broiler chickens. Poult. Sci.  91: 2255– 2263. Google Scholar CrossRef Search ADS PubMed  © 2018 Poultry Science Association Inc. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Evaluation of dietary calcium level and source and phytase on growth performance, serum metabolites, and ileum mineral contents in broiler chicks fed adequate phosphorus diets from one to 28 days of age

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Oxford University Press
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© 2018 Poultry Science Association Inc.
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0032-5791
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1525-3171
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Abstract

Abstract A total of 936 one-day-old broiler chicks (Ross 308) was used to evaluate the effects of dietary calcium (Ca) source (1.0 and 0.6% from Ca carbonate [CC], or 0.6% from Celtic sea minerals [CSM]) on broiler chick response to phytase supplementation (0, 500, or 2,500 FTU per kg of diet) in phosphorus (P) adequate diets. Birds were randomly assigned to 9 treatments, each replicated 8 times (4 repeats of male and 4 repeats of female birds; 13 birds per replicate) in a completely randomized design. Results showed that birds fed low Ca CC diets had significantly (P ≤ 0.05) lower body weight at 14 and 21 d; weight gain during 1 to 14 d; feed intake during 1 to 14 d and 1 to 28 d; and toe and tibia ash content, ileum ash, and Ca, and P contents at 28 days. Feed conversion ratio and feed intake between 21 and 28 d, and serum Ca, Fe, and alkaline phosphatase levels at 28 d of age were not affected by dietary Ca level. The negative influence of reducing the dietary Ca level on body weight and weight gain was less severe when CC was replaced with CSM, and birds fed the CSM diet had a significantly lower feed conversion ratio and higher tibia P content compared to CC. Phytase did not have a significant influence (P > 0.05) on most measured parameters, but significantly reduced ileum P and ash contents, especially at the higher level of 2,500 vs. 500 units of phytase per kg of diet. These results confirm that CSM has a valuable potential to improve feed conversion ratio, and that phytase was less effective in compensating for a significantly reduced dietary Ca level as it is for P. INTRODUCTION It has been well documented that it is not only important to meet the dietary calcium (Ca) and phosphorus (P) requirements in broiler diet formulation, but also to target the appropriate ratio of Ca: P in order to ensure maximum performance and bone mineralization (Plumstead et al., 2008; Delezie et al., 2012; Dos Santos et al., 2013). Although the broiler chick needs more Ca than P, the higher cost of meeting its P demands and environmental concern relating to excess excreta P has motivated much more work on P than Ca. Such work has focused on the evaluation of different organic and inorganic P sources in order to determine their availability and at the same time the P requirement of the broiler. Work also has considered methods to improve the broilers’ ability to utilize plant derived P source via exogenous phytase (Selle and Ravindran, 2007; Plumstead et al., 2008; Delezie et al., 2012). However, the broiler chicks’ response to phytase is not straightforward, as it is influenced by many factors (Sebastian et al., 1998; Selle and Ravindran, 2007). Dietary Ca level is one of the most relevant feed items, as it not only markedly influences the broiler chicks’ P requirements, but also affects phytase efficacy (Dos Santos et al., 2013). Although some work has focused on Ca requirements, less work has been done to investigate the impact of dietary Ca source on phytase efficacy or explore the potential of phytase to enhance dietary Ca retention in low Ca diets (Augspurger and Baker, 2004; Dos Santos et al., 2013). Celtic sea mineral (CSM) is a calcareous marine algae (Lithothamnion calcareum), highly calcified with a mix of calcite, aragonite, and vaterite (Schlegel and Gutzwiller, 2016) and is an alternative inorganic source of Ca (up to 32% Ca). Though the Ca is the major mineral component of CSM, it also contains other minerals, such as Mg, Fe, P, Mn, I, Zn, etc. (González-Vega et al., 2014). Although many studies addressed the nutritional value of Ca carbonate, few studies reported the potential of CSM and its interaction with exogenous phytase in broiler chicks’ nutrition (González-Vega et al., 2014; Schlegel and Gutzwiller, 2016). Therefore, the aim of the present study was to determine the effects of different dietary Ca sources (Ca carbonate [CC] or CSM) on phytase efficacy in broiler chicks fed a P adequate diet. MATERIAL AND METHODS Diet Preparation Dietary treatments included a corn-soybean-meal-based diet (Table 1) with the recommended level of dietary Ca (1.0% Ca from CC; 0.6% from CC, and 0.6% of CSM source) in non-phytate P (nPP) adequate diets (0.50%). Each diet was supplemented with a graded level of phytase (0, 500, and 2,500 FTU per kg of the diet; Quantum phytase 5000 XT, AB-Vista Feed Ingredients, Marlborough, Wiltshire SN8 4AN, United Kingdom). The phytase supplement was a preparation of an evolved Escherichia coli 6-phytase (EC 3.1.2.26; 5,000 FTU g−1), produced by the genetically modified yeast Pichia pastoris. One unit of phytase activity was defined as the amount of enzyme that liberates one micromole of inorganic phosphate from sodium phytate at pH 5.5 and 37°C (Table 2). All enzymes were added to the representative basal diets as granules. To achieve maximum mixing uniformity, the enzyme was first mixed with a small quantity of each complete diet prior to incorporation into the mix with the rest of the diet. The CSM Ca source was obtained from Celtic Sea Minerals, Currabinny, Co Cork, Ireland, and is derived from Lithothmnion calcareum, a calcified sea weed derived product. Chicks and Rearing In this experiment, 936 one-day-old broiler chicks (Ross 308) were obtained from a local hatchery and randomly distributed among 9 treatments, each replicated 8 times (4 male replicates and 4 female replicates; each replicate contained 13 birds). The experiment lasted 28 days. All chicks were housed in 72 floor pens (1.25 × 1.4 m), with wood shavings used as litter. Birds received continuous lighting during the first 24 h, then maintained on a 23 L: 1D schedule during the rest of the experimental period. The experimental house temperature was maintained between 30 and 32°C at the beginning of the experiment and then gradually decreased by 2 to 3°C each wk to reach a final temperature of 22°C at the end. Chicks had free access to the mash diet and fresh water during the experiment. Care and management of the chicks were in accordance with commercial guidelines and were approved by University of Kurdistan Animal Ethics Committee. Diets Composition As described in Table 1, basal corn-soybean meal diets were formulated to meet or exceed the nutritional requirements of broiler chicks (NRC, 1994) for all nutrients with the exception of Ca. The experimental diets were formulated to contain different Ca levels derived from 2 different Ca sources, CC and CSM. Dietary Ca levels investigated were 1% from CC, 0.6% from CC, and 0.6% from CSM. Basal diets were formulated to be isonitrogenous, isocaloric, and isophosphoric (0.50% nPP). The nutritional composition of experimental diets are shown in Table 1. Table 1. Composition (%) and calculated nutrient content of experimental diets.   High calcium  Low calcium    Calcium  Calcium  Celtic sea    carbonate  carbonate  mineral  Ingredient  (CC)  (CC)  (CSM)  Corn  58.95  61.04  60.57  Soybean meal 46  33.18  32.83  32.91  Soy oil  2.79  2.09  2.25  Salt  0.28  0.28  0.28  DL Methionine  0.32  0.32  0.32  Lysine HCl  0.27  0.28  0.27  Threonine  0.07  0.07  0.07  Calcium carbonate1  1.97  0.92  0.00  Dicalcium Phosphate  0.65  0.65  0.65  Celtic sea mineral2  0.00  0.00  1.16  Phosphoric acid  1.00  1.00  1.00  Coccidiostat (Coban - monensin)  0.02  0.02  0.02  Vitamin premix3  0.25  0.25  0.25  Mineral premix4  0.25  0.25  0.25  Calculated analysis  Crude protein %  21.40  21.40  21.40  Poult ME kcal/kg  3085  3085  3085  Calcium %  1.00  0.60  0.60  Phos %  0.79  0.79  0.79  Avail phos %  0.50  0.50  0.50  Arg %  1.41  1.41  1.41  Cysteine, %  0.35  0.35  0.35  Lysine, %  1.35  1.35  1.35  Methionine, %  0.65  0.65  0.65  Total sulphur amino acids, %  1.00  1.00  1.00  Tryp %  0.24  0.24  0.24  Na %  0.18  0.18  0.18  Cl %  0.26  0.26  0.26  K %  0.91  0.90  0.90  Analyzed nutrients, %  Dry matter, %  89.66  89.54  89.52  Ash, %  5.68  5.35  5.26  Crude protein, %  19.67  19.66  20.05  Ca, %  1.15  0.77  0.75  Total P,%  0.73  0.70  0.74    High calcium  Low calcium    Calcium  Calcium  Celtic sea    carbonate  carbonate  mineral  Ingredient  (CC)  (CC)  (CSM)  Corn  58.95  61.04  60.57  Soybean meal 46  33.18  32.83  32.91  Soy oil  2.79  2.09  2.25  Salt  0.28  0.28  0.28  DL Methionine  0.32  0.32  0.32  Lysine HCl  0.27  0.28  0.27  Threonine  0.07  0.07  0.07  Calcium carbonate1  1.97  0.92  0.00  Dicalcium Phosphate  0.65  0.65  0.65  Celtic sea mineral2  0.00  0.00  1.16  Phosphoric acid  1.00  1.00  1.00  Coccidiostat (Coban - monensin)  0.02  0.02  0.02  Vitamin premix3  0.25  0.25  0.25  Mineral premix4  0.25  0.25  0.25  Calculated analysis  Crude protein %  21.40  21.40  21.40  Poult ME kcal/kg  3085  3085  3085  Calcium %  1.00  0.60  0.60  Phos %  0.79  0.79  0.79  Avail phos %  0.50  0.50  0.50  Arg %  1.41  1.41  1.41  Cysteine, %  0.35  0.35  0.35  Lysine, %  1.35  1.35  1.35  Methionine, %  0.65  0.65  0.65  Total sulphur amino acids, %  1.00  1.00  1.00  Tryp %  0.24  0.24  0.24  Na %  0.18  0.18  0.18  Cl %  0.26  0.26  0.26  K %  0.91  0.90  0.90  Analyzed nutrients, %  Dry matter, %  89.66  89.54  89.52  Ash, %  5.68  5.35  5.26  Crude protein, %  19.67  19.66  20.05  Ca, %  1.15  0.77  0.75  Total P,%  0.73  0.70  0.74  1Calcium carbonate, 38% Ca. 2Celtic sea mineral, 32.5% Ca. 3Provides per kg of diet: Vit. A (as all-trans retinol acetate), 9,000 I.U.; Cholecalciferol, 2,000 I.U.; Vit. E (as dl- alpha-tocopheryl acetate), 18 I.U.; Vit K (as menadion sodium bisulfate), 2 mg; Thiamine (as thiamin mononitrate), 1.8 mg; Riboflavin, 6.6 mg; Niacin, 30 mg; Pyridoxin, 3 mg; Vit B12, 15 mcg; Calcium d-Pantothenate, 10 mg; Folic acid, 1 mg; Biotin (as d-biotin), 0.1 mg; Choline chloride (as choline chloride), 500 mg; Antioxidant (as butylatedhydroxy toluene), 100 mg. 4Provides per Kg of diet: Manganese (as MnO), 100 mg; Zinc (ZnSO4. 7H2O), 84.7 mg; Iron (FeSO4. 7H2O), 50 mg; Copper (CuSO4. 5H2O), 10 mg; Iodine (KI), 1 mg; Se (Na2SeO3), 0.2 mg. View Large Table 2. Phytase activity in experimental diets.1 Dietary treatments  Phytase recovery  Ca Source  Ca Level  Phytase  (FTU. Kg−1)    (%)  (U.Kg−1)    CC  1.0  0  <50  CC  1.0  500  526  CC  1.0  2,500  2,263  CC  0.6  0  ∼93  CC  0.6  500  711  CC  0.6  2,500  2,257  CSM  0.6  0  ∼72  CSM  0.6  500  543  CSM  0.6  2,500  2,548  Dietary treatments  Phytase recovery  Ca Source  Ca Level  Phytase  (FTU. Kg−1)    (%)  (U.Kg−1)    CC  1.0  0  <50  CC  1.0  500  526  CC  1.0  2,500  2,263  CC  0.6  0  ∼93  CC  0.6  500  711  CC  0.6  2,500  2,257  CSM  0.6  0  ∼72  CSM  0.6  500  543  CSM  0.6  2,500  2,548  1Dietary phytase quantification determined using ELISA method by AB Vista on final diets (Engelen et al., 2001). CSM: Celtic sea minerals, CC: Calcium carbonate. View Large Measurements Birds were weighed as a group on arrival and at 14, 21, and 28 d of age on a pen basis. Feed intake was also recorded at the same time points for calculation of feed conversion ratio after adjustment for the weight of dead birds in each growth period. Average body weight, body weight gain, feed intake, and feed conversion ratio were determined between 1 to 14, 14 to 21, 21 to 28, and 1 to 28 d of age. At 28 d of age, 3 birds per pen were randomly selected for blood sampling from the left wing vein. After sampling, these selected birds were killed by cervical dislocation, and the right and left tibia and all toe bones were removed. Subsequently, tibias were de-fleshed and defatted in a mixture of 90% ethyl ether and 10% methanol. Finally, pooled toe and defatted tibia bones were dried at 105°C until a consistent weight was obtained, and then ashed in a muffled furnace at 605°C for at least 12 h (AOAC, 1990). The ash content was expressed as g of ash per 100 g of the dried weight for each bone. At the same time, ileum digesta samples from all sacrificed birds were collected for ash, Ca, and P analysis. The Ca and P contents of dried tibia and ileum digesta were measured (AOAC, 1990). Blood serum P, Ca, Fe, and alkaline phosphatase were measured using enzymatic, colorimetric essays using the relevant clinical kits (Pars Azmun, Tehran, Iran). The chemical composition of experimental diets, including DM, CP, Ca, P (AOAC, 1990), and phytase (Engelen et al., 2001), also were measured as described in Table 1. Statistical Analysis Data were analyzed using the General Linear Models (GLM) procedure of SAS (SAS institute, 1991) using a completely randomized design (CRD) in a factorial arrangement. Mortality data were transformed using $$\sqrt {X + 1}$$ prior to analysis (Manikandan, 2010). Significant differences among treatments were determined at P ≤ 0.05 using Tukey tests. Additional analyses were conducted using contrast statements to examine the effects of Ca source at 0.6% Ca inclusion (CC vs. CSM) or Ca level (1.0 vs. 0.6%). The dose-response effect of supplemental phytase was computed using orthogonal polynomial contrast for liner and quadratic effects (SAS, 1991). RESULTS Performance No significant interaction effects of treatment on performance were noted when the whole data set was considered, but in contrast, comparing Ca levels between the 2 CC diets, it was clear that the lower level (0.6%) led to significantly lower weight gain (1 to 14, 14 to 21, and 1 to 28 d) and body weights (14, 21, and 28 d) compared with the 1% control. No differences between calcium sources or phytase levels were found (P > 0.05) on body weight or gain (Table 3). Table 3. Effects of calcium source, level, and phytase supplementation on body weight and weight gain (g) in broiler chicks fed high phosphorus diets from 1 to 28 d of age.     Body weight (g)  Weight gain (g)      1 d  14 d  21 d  28 d  1 to 14 d  14 to 21 d  21 to 28 d  1 to 28 d  Ca Source   CC, 1.0% Ca    44  407a  828a  1313  363a  421  485  1269   CC, 0.60% Ca    45  389b  787b  1245  345b  398  458  1201   CSM, 0.60% Ca    44  400a,b  810a,b  1288  355a,b  411  478  1244  Phytase level (U. Kg−1)   0.0    44  394  804  1278  350  410  475  1234   500    44  404  816  1293  360  412  477  1248   2,500    44  398  806  1275  354  409  469  1231  Ca source × Phytase level   CC, 1.0% Ca  0  45  412  843  1325  368  430  483  1281    500  44  414  831  1327  370  417  496  1283    2,500  44  396  811  1287  352  415  476  1243   CC, 0.6% Ca  0  44  381  776  1232  337  395  457  1188    500  44  401  817  1289  357  416  472  1245    2,500  44  384  768  1213  340  384  445  1169   CSM, 0.6% Ca  0  44  389  793  1278  345  404  485  1233    500  44  397  798  1261  353  401  463  1217    2,500  44  413  840  1326  369  427  486  1281   SEM    0.22  9.08  20.23  36.67  9.03  12.70  19.84  36.62   Ca source (Ca)    0.574  0.046  0.049  0.077  0.047  0.099  0.231  0.077   Phytase level (P)    0.307  0.401  0.750  0.828  0.380  0.961  0.879  0.825   Ca × P    0.605  0.150  0.136  0.369  0.144  0.176  0.725  0.366  Source of variation of phytase level, P-value   Linear    0.150  0.183  0.471  0.634  0.170  0.847  0.886  0.628   Quadratic    0.593  0.832  0.823  0.700  0.840  0.838  0.628  0.702  Orthogonal contrasts   CC, 1.0% vs. CC, 0.6%    0.406  0.014  0.015  0.026  0.014  0.033  0.100  0.026   CC, 0.6% vs. CSM, 0.6%    0.331  0.144  0.163  0.153  0.149  0.238  0.222  0.154   CC, 1.0% vs. CSM, 0.6%    0.888  0.300  0.277  0.404  0.293  0.324  0.665  0.403      Body weight (g)  Weight gain (g)      1 d  14 d  21 d  28 d  1 to 14 d  14 to 21 d  21 to 28 d  1 to 28 d  Ca Source   CC, 1.0% Ca    44  407a  828a  1313  363a  421  485  1269   CC, 0.60% Ca    45  389b  787b  1245  345b  398  458  1201   CSM, 0.60% Ca    44  400a,b  810a,b  1288  355a,b  411  478  1244  Phytase level (U. Kg−1)   0.0    44  394  804  1278  350  410  475  1234   500    44  404  816  1293  360  412  477  1248   2,500    44  398  806  1275  354  409  469  1231  Ca source × Phytase level   CC, 1.0% Ca  0  45  412  843  1325  368  430  483  1281    500  44  414  831  1327  370  417  496  1283    2,500  44  396  811  1287  352  415  476  1243   CC, 0.6% Ca  0  44  381  776  1232  337  395  457  1188    500  44  401  817  1289  357  416  472  1245    2,500  44  384  768  1213  340  384  445  1169   CSM, 0.6% Ca  0  44  389  793  1278  345  404  485  1233    500  44  397  798  1261  353  401  463  1217    2,500  44  413  840  1326  369  427  486  1281   SEM    0.22  9.08  20.23  36.67  9.03  12.70  19.84  36.62   Ca source (Ca)    0.574  0.046  0.049  0.077  0.047  0.099  0.231  0.077   Phytase level (P)    0.307  0.401  0.750  0.828  0.380  0.961  0.879  0.825   Ca × P    0.605  0.150  0.136  0.369  0.144  0.176  0.725  0.366  Source of variation of phytase level, P-value   Linear    0.150  0.183  0.471  0.634  0.170  0.847  0.886  0.628   Quadratic    0.593  0.832  0.823  0.700  0.840  0.838  0.628  0.702  Orthogonal contrasts   CC, 1.0% vs. CC, 0.6%    0.406  0.014  0.015  0.026  0.014  0.033  0.100  0.026   CC, 0.6% vs. CSM, 0.6%    0.331  0.144  0.163  0.153  0.149  0.238  0.222  0.154   CC, 1.0% vs. CSM, 0.6%    0.888  0.300  0.277  0.404  0.293  0.324  0.665  0.403  a,bMeans in column and under each main effects with common superscript do not differ significantly (P ≤ 0.05). CSM: Celtic sea minerals, CC: Calcium carbonate. View Large Treatment effects on feed intake were noted between 1 to 14, 14 to 21, and 1 to 28 d of age only (P ≤ 0.05), Reducing dietary Ca level from 1.0 to 0.6% reduced (P ≤ 0.05) feed intake between 1 to 14 (538 vs. 572 g), 14 to 21 (657 vs. 690), and 1 to 28 (2,057 vs. 2,156) d but not during the last wk (21 to 28 d). Phytase did not influence feed intake during any period (P > 0.05). Treatment effects on feed conversion were detected between 14 to 21 and 1 to 28 d of age (P ≤ 0.05). The contrast data suggest that FCR was significantly better for birds fed the CSM diet compared with the same amount of Ca derived from the CC diet (P ≤ 0.05). Feed conversion ratio was not influenced by the addition of phytase. As presented in Table 3 and Table 4, addition of different levels of phytase had no linear or quadratic effects on performance criteria. Mortality (%) was not influenced by dietary Ca level (1.0 vs. 0.6%) or Ca source (data not shown). Table 4. Effects of calcium source level and phytase supplementation on feed intake (g) and feed conversion ratio (g. g−1) in broiler chicks fed a high phosphorus diet from 1 to 28 d of age.     Feed intake (g)  Feed conversion ratio (g. g−1)      1 to 14 d  14 to 21 d  21 to 28 d  1 to 28 d  1 to 14 d  14 to 21 d  21 to 28 d  1 to 28 d  Ca Source   CC, 1.0% Ca    572a  690a  905  2156a  1.58  1.64a,b  1.88  1.70a   CC, 0.60% Ca    538b  657a,b  873  2057b  1.56  1.66a  1.92  1.72a   CSM, 0.60% Ca    538b  651b  876  2052b  1.52  1.59b  1.84  1.65b  Phytase level (U. Kg−1)   0.0    546  669  877  2080  1.56  1.64  1.86  1.69   500    556  672  900  2115  1.54  1.64  1.90  1.70   2,500    546  658  877  2070  1.56  1.62  1.88  1.69  Ca source × Phytase level   CC, 1.0% Ca  0  568  699  910  2165  1.55  1.62  1.89  1.69    500  574  685  931  2177  1.55  1.65  1.88  1.70    2,500  573  686  873  2126  1.64  1.65  1.86  1.71   CC, 0.6% Ca  0  534  658  855  2037  1.58  1.67  1.88  1.72    500  563  681  913  2145  1.56  1.64  1.95  1.73    2,500  517  633  852  1989  1.54  1.66  1.93  1.71   CSM, 0.6% Ca  0  535  649  866  2038  1.56  1.61  1.80  1.66    500  531  649  856  2024  1.50  1.62  1.86  1.67    2,500  548  656  906  2095  1.50  1.54  1.87  1.64   SEM    11.93  17.48  26.15  46.91  0.03  0.03  0.05  0.02   Ca source (Ca)    0.001  0.019  0.270  0.013  0.115  0.030  0.166  0.004   Phytase level (P)    0.511  0.612  0.458  0.465  0.634  0.740  0.625  0.857   Ca × P    0.135  0.506  0.183  0.199  0.193  0.354  0.861  0.870  Source of variation of phytase level, P-value   Linear    0.305  0.818  0.278  0.360  0.374  0.966  0.344  0.679   Quadratic    0.593  0.337  0.537  0.405  0.732  0.440  0.850  0.714  Orthogonal contrasts   CC, 1.0% vs. CC, 0.6%    0.001  0.026  0.146  0.012  0.459  0.568  0.330  0.416   CC, 0.6% vs. CSM, 0.6%    0.978  0.682  0.902  0.909  0.184  0.012  0.059  0.001   CC, 1.0% vs. CSM, 0.6%    0.001  0.009  0.182  0.009  0.041  0.048  0.351  0.014      Feed intake (g)  Feed conversion ratio (g. g−1)      1 to 14 d  14 to 21 d  21 to 28 d  1 to 28 d  1 to 14 d  14 to 21 d  21 to 28 d  1 to 28 d  Ca Source   CC, 1.0% Ca    572a  690a  905  2156a  1.58  1.64a,b  1.88  1.70a   CC, 0.60% Ca    538b  657a,b  873  2057b  1.56  1.66a  1.92  1.72a   CSM, 0.60% Ca    538b  651b  876  2052b  1.52  1.59b  1.84  1.65b  Phytase level (U. Kg−1)   0.0    546  669  877  2080  1.56  1.64  1.86  1.69   500    556  672  900  2115  1.54  1.64  1.90  1.70   2,500    546  658  877  2070  1.56  1.62  1.88  1.69  Ca source × Phytase level   CC, 1.0% Ca  0  568  699  910  2165  1.55  1.62  1.89  1.69    500  574  685  931  2177  1.55  1.65  1.88  1.70    2,500  573  686  873  2126  1.64  1.65  1.86  1.71   CC, 0.6% Ca  0  534  658  855  2037  1.58  1.67  1.88  1.72    500  563  681  913  2145  1.56  1.64  1.95  1.73    2,500  517  633  852  1989  1.54  1.66  1.93  1.71   CSM, 0.6% Ca  0  535  649  866  2038  1.56  1.61  1.80  1.66    500  531  649  856  2024  1.50  1.62  1.86  1.67    2,500  548  656  906  2095  1.50  1.54  1.87  1.64   SEM    11.93  17.48  26.15  46.91  0.03  0.03  0.05  0.02   Ca source (Ca)    0.001  0.019  0.270  0.013  0.115  0.030  0.166  0.004   Phytase level (P)    0.511  0.612  0.458  0.465  0.634  0.740  0.625  0.857   Ca × P    0.135  0.506  0.183  0.199  0.193  0.354  0.861  0.870  Source of variation of phytase level, P-value   Linear    0.305  0.818  0.278  0.360  0.374  0.966  0.344  0.679   Quadratic    0.593  0.337  0.537  0.405  0.732  0.440  0.850  0.714  Orthogonal contrasts   CC, 1.0% vs. CC, 0.6%    0.001  0.026  0.146  0.012  0.459  0.568  0.330  0.416   CC, 0.6% vs. CSM, 0.6%    0.978  0.682  0.902  0.909  0.184  0.012  0.059  0.001   CC, 1.0% vs. CSM, 0.6%    0.001  0.009  0.182  0.009  0.041  0.048  0.351  0.014  a,bMeans in column and under each main effects with common superscript do not differ significantly (P ≤ 0.05). CSM: Celtic sea minerals, CC: Calcium carbonate. View Large Bone Mineralization No significant treatment effects were noted for tibia ash, although the results suggested there was a significant reduction in toe ash when (P ≤ 0.05) the dietary Ca level was reduced from 1.0 to 0.6%. Phytase supplementation had no significant (P > 0.05) effect on toe ash content (P > 0.05). As shown in Table 5, though tibia ash content (%) at 28 d of age was not influenced by treatment, the contrast of the low vs. high CC Ca level suggested the latter had greater ash content (P ≤ 0.05). Phytase had no effect, and there were no differences (P > 0.05) between the 2 Ca sources noted for tibia ash (Table 5). Table 5. Effects of calcium source level and phytase supplementation on broiler chicks bone mineralization and ileum mineral contents at 28 d of age.     Toe Ash (%)  Tibia contents (%)  Ileum content (%)        Ash  Ca  P  Ash  Ca  P  Ca Source   CC, 1.0% Ca    14.3a  37.8  10.3b  6.1a,b  11.7a  0.47a  1.14a   CC, 0.60% Ca    13.2b  36.7  11.3a  5.9b  8.9b  0.27b  0.88b   CSM, 0.60% Ca    13.3b  37.5  11.4a  6.2a  8.9b  0.29b  0.85b  Phytase level (U. Kg−1)   0.0    13.8  37.5  11.5  6.1  10.4a  0.34  1.07a   500    14.0  37.1  10.3  6.0  10.0a  0.35  0.96b   2,500    13.1  37.4  11.2  6.1  9.2b  0.34  0.85c  Ca source × Phytase level   CC, 1.0% Ca  0  15.4  38.1  10.7a,b  6.1  12.0  0.51  1.18a,b    500  14.1  37.5  9.0b  6.1  12.4  0.47  1.21a    2,500  13.5  37.9  11.1a,b  6.0  10.7  0.44  1.04a–c   CC, 0.6% Ca  0  13.1  37.4  12.7a  6.0  9.2  0.25  0.98b–d    500  13.9  36.3  10.9a,b  5.9  9.1  0.28  0.87d,e    2,500  12.8  36.4  10.3a,b  5.7  8.1  0.26  0.79d,e   CSM, 0.6% Ca  0  13.1  36.9  11.1a,b  6.1  9.4  0.27  1.03a–c    500  13.9  37.7  11.0a,b  6.0  8.6  0.29  0.79d,e    2,500  13.0  37.8  12.1a  6.5  8.7  0.31  0.73e   SEM                   Ca source (Ca)    0.046  0.099  0.045  0.013  <0.0001  <0.0001  <0.0001   Phytase level (P)    0.156  0.820  0.059  0.773  0.001  0.920  <0.0001   Ca × P    0.323  0.556  0.028  0.065  0.218  0.424  <0.048  Source of variation of phytase level, P-value   Linear    0.777  0.544  0.024  0.582  0.338  0.817  <0.006   Quadratic    0.057  0.870  0.477  0.653  0.0003  0.740  <0.0001  Orthogonal contrasts   CC, 1.0% vs. CC, 0.6%    0.023  0.037  0.042  0.047  <0.0001  <0.0001  <0.0001   CC, 0.6% vs. CSM, 0.6%    0.855  0.136  0.782  0.004  0.995  0.275  0.497   CC, 1.0% vs. CSM, 0.6%    0.043  0.540  0.024  0.355  <0.0001  <0.0001  <0.0001      Toe Ash (%)  Tibia contents (%)  Ileum content (%)        Ash  Ca  P  Ash  Ca  P  Ca Source   CC, 1.0% Ca    14.3a  37.8  10.3b  6.1a,b  11.7a  0.47a  1.14a   CC, 0.60% Ca    13.2b  36.7  11.3a  5.9b  8.9b  0.27b  0.88b   CSM, 0.60% Ca    13.3b  37.5  11.4a  6.2a  8.9b  0.29b  0.85b  Phytase level (U. Kg−1)   0.0    13.8  37.5  11.5  6.1  10.4a  0.34  1.07a   500    14.0  37.1  10.3  6.0  10.0a  0.35  0.96b   2,500    13.1  37.4  11.2  6.1  9.2b  0.34  0.85c  Ca source × Phytase level   CC, 1.0% Ca  0  15.4  38.1  10.7a,b  6.1  12.0  0.51  1.18a,b    500  14.1  37.5  9.0b  6.1  12.4  0.47  1.21a    2,500  13.5  37.9  11.1a,b  6.0  10.7  0.44  1.04a–c   CC, 0.6% Ca  0  13.1  37.4  12.7a  6.0  9.2  0.25  0.98b–d    500  13.9  36.3  10.9a,b  5.9  9.1  0.28  0.87d,e    2,500  12.8  36.4  10.3a,b  5.7  8.1  0.26  0.79d,e   CSM, 0.6% Ca  0  13.1  36.9  11.1a,b  6.1  9.4  0.27  1.03a–c    500  13.9  37.7  11.0a,b  6.0  8.6  0.29  0.79d,e    2,500  13.0  37.8  12.1a  6.5  8.7  0.31  0.73e   SEM                   Ca source (Ca)    0.046  0.099  0.045  0.013  <0.0001  <0.0001  <0.0001   Phytase level (P)    0.156  0.820  0.059  0.773  0.001  0.920  <0.0001   Ca × P    0.323  0.556  0.028  0.065  0.218  0.424  <0.048  Source of variation of phytase level, P-value   Linear    0.777  0.544  0.024  0.582  0.338  0.817  <0.006   Quadratic    0.057  0.870  0.477  0.653  0.0003  0.740  <0.0001  Orthogonal contrasts   CC, 1.0% vs. CC, 0.6%    0.023  0.037  0.042  0.047  <0.0001  <0.0001  <0.0001   CC, 0.6% vs. CSM, 0.6%    0.855  0.136  0.782  0.004  0.995  0.275  0.497   CC, 1.0% vs. CSM, 0.6%    0.043  0.540  0.024  0.355  <0.0001  <0.0001  <0.0001  a–eMeans in column and under each main effects with common superscript do not differ significantly (P ≤ 0.05). CSM: Celtic sea minerals, CC: Calcium carbonate. View Large Tibia Ca content (%) was significantly influenced by dietary Ca source × phytase level interaction (P < 0.05), which seemed to be due to lower levels being found in the 500 FTU CC 1% diet compared with the unsupplemented CC 0.6% diet and the 2,500 FTU supplemented CSM diet. Tibia P was minimized in the lower Ca CC diets. As presented in Tables 3 and 4, supplementation of different levels of phytase to the basal diet had no linear or quadratic effect. Mineral Content of the Ileum Ileal content of ash, Ca, and P was significantly influenced by reduction of dietary Ca level, regardless of Ca source. In addition, phytase supplementation reduced ash and P contents of the ileum, particularly at the highest dose in the CSM diet. As noted in Table 5, addition of graded levels of phytase to the basal diet resulted in linear and quadratic reduction in ileum P content. Serum Metabolites As shown in Table 6, there were no significant treatment effects on serum traits at 28 d of age, although the effect of Ca level reduction on serum P was significant. Serum P was lower in birds fed the high Ca CC diets compared with the low Ca CC diets. No effects of phytase on serum traits were noted (P > 0.05). Table 6. Effects of calcium source level and phytase supplementation on blood serum components of broiler chicks at 28 d of age.     Calcium (mg/ dl)  Fe (μg/dl)  Alkaline phosphatase (IU/l)  Phosphorus (mg/dl)  Ca Source   CC, 1.0% Ca    8.8  89.2  2906  5.9b   CC, 0.60% Ca    8.7  87.4  3129  6.5a,b   CSM, 0.60% Ca    9.0  95.1  2860  6.6a  Phytase level (U. Kg−1)   0.0    9.1  88.4  2660  6.2   500    8.9  95.4  3062  6.2   2,500    8.4  88.1  3217  6.5  Ca source × Phytase level   CC, 1.0% Ca  0  9.2  83.3  2654.8  6.1    500  9.1  103.8  2688.0  5.9    2,500  8.1  77.3  3338.2  5.8   CC, 0.6% Ca  0  9.1  81.8  2845.1  6.1    500  8.9  95.9  3576.7  6.5    2,500  8.2  86.4  3076.3  6.8   CSM, 0.6% Ca  0  9.2  101.3  2455.6  6.4    500  8.7  87.7  2920.5  6.3    2,500  9.1  97.3  3213.6  7.1   SEM      0.41  10.05  451.16   Ca source (Ca)    0.715  0.572  0.672  0.019   Phytase level (P)    0.127  0.528  0.260  0.271   Ca × P    0.459  0.317  0.763  0.248  Source of variation of phytase level, P-value   Linear    0.506  0.376  0.277  0.944   Quadratic    0.055  0.477  0.249  0.108  Orthogonal contrasts   CC, 1.0% vs. CC, 0.6%    0.896  0.998  0.482  0.049   CC, 0.6% vs. CSM, 0.6%    0.446  0.363  0.415  0.435   CC, 1.0% vs. CSM, 0.6%    0.521  0.371  0.905  0.007      Calcium (mg/ dl)  Fe (μg/dl)  Alkaline phosphatase (IU/l)  Phosphorus (mg/dl)  Ca Source   CC, 1.0% Ca    8.8  89.2  2906  5.9b   CC, 0.60% Ca    8.7  87.4  3129  6.5a,b   CSM, 0.60% Ca    9.0  95.1  2860  6.6a  Phytase level (U. Kg−1)   0.0    9.1  88.4  2660  6.2   500    8.9  95.4  3062  6.2   2,500    8.4  88.1  3217  6.5  Ca source × Phytase level   CC, 1.0% Ca  0  9.2  83.3  2654.8  6.1    500  9.1  103.8  2688.0  5.9    2,500  8.1  77.3  3338.2  5.8   CC, 0.6% Ca  0  9.1  81.8  2845.1  6.1    500  8.9  95.9  3576.7  6.5    2,500  8.2  86.4  3076.3  6.8   CSM, 0.6% Ca  0  9.2  101.3  2455.6  6.4    500  8.7  87.7  2920.5  6.3    2,500  9.1  97.3  3213.6  7.1   SEM      0.41  10.05  451.16   Ca source (Ca)    0.715  0.572  0.672  0.019   Phytase level (P)    0.127  0.528  0.260  0.271   Ca × P    0.459  0.317  0.763  0.248  Source of variation of phytase level, P-value   Linear    0.506  0.376  0.277  0.944   Quadratic    0.055  0.477  0.249  0.108  Orthogonal contrasts   CC, 1.0% vs. CC, 0.6%    0.896  0.998  0.482  0.049   CC, 0.6% vs. CSM, 0.6%    0.446  0.363  0.415  0.435   CC, 1.0% vs. CSM, 0.6%    0.521  0.371  0.905  0.007  a,bMeans in column and under each main effects with common superscript do not differ significantly (P ≤ 0.05). CSM: Celtic sea minerals, CC: Calcium carbonate. View Large DISCUSSION The findings of the current experiment with regard to the negative consequences of reducing dietary Ca from 1.0 to 0.6% in a P adequate diet (0.5%) on body weight, weight gain, feed intake, toe and tibia ash contents are in agreement with previously published reports (Driver et al., 2005; Delezie et al., 2012). These data indicate that the Ca deficiency in the current experiment when CC was fed at 0.6% was moderate and likely a result of the fact that the P levels were simultaneously luxurious. The analyzed Ca content of the low Ca diets was approximately 0.77%, which would be deemed adequate if the available phosphate levels were approximately 0.35%, but in this study they were formulated to be much higher at 0.5%. In addition, the data confirm that some criteria, such as body weight, are more responsive to dietary Ca level than feed conversion ratio and serum mineral contents (Dos Santos et al., 2013). Driver et al. (2005) demonstrated that reducing dietary Ca level to less than 0.625% in a high P diet (0.45%) had negative effects on body weight to 16 d of age, but in lower P diets, this detrimental effect was reduced. Augspurger and Baker (2004) also showed that reducing dietary Ca level from 1 to 0.48% in a high nPP diet (0.45%) significantly reduced weight gain. It has been postulated that the negative impacts of reducing dietary Ca level on broiler performance are just as much related to disturbing the dietary Ca: available P balance as it is to shortage of Ca supply. Thus, the effects of feeding a particular dietary Ca level needs to take into account the P level of the diet, as this dictates whether an appropriate Ca: available P ratio has been achieved (Shafey, 1993; Augspurger and Baker, 2004). Some researchers suggest that the absolute Ca and P requirements of broiler chicks is less than the current NRC (1994) recommendation or broiler company nutrient specifications. As a result, the observed adverse effects noted in some research of feeding low Ca diets to young broiler chicks is more of a consequence of a narrow dietary Ca: P balance rather than a Ca deficiency per se (Delezie et al., 2012). Delezie et al. (2012) reported that concomitant and coordinated reduction of dietary Ca and P levels had no deleterious effects on bone mineralization, but feeding an imbalanced Ca and P diet significantly reduced body weight, bone development, and Ca and P retention. The current findings that CSM was better than CC as a Ca source in a low Ca diet, with lower feed conversion ratios, is partially explained by higher solubility of CSM compared with CC (Walk et al., 2012). This would contribute to correcting the Ca: P ratio imbalance caused by feeding a low Ca, high P level in the basal diet. Use of a dietary Ca source such as CSM, which has higher Ca solubility than commonly used Ca sources such as CC, will enable the use of lower Ca diets without imbalancing the dietary Ca: P ratio. There were few if any beneficial effects of feeding phytase on performance or tibia ash content (%) in this study. Use of this phytase at 500 FTU would be expected to release Ca and P in an approximate 1:1 ratio, which, in the low Ca diets, would further imbalance the Ca: P ratio. Even though the amount of digestible Ca would be increased, this benefit is overshadowed by the negative effect of the incremental P digestibility on the ratio between Ca and P. Indeed, phytase supplementation, especially at the highest level, significantly reduced ileum ash and P content, suggesting better absorption, but it was less effective on ileum Ca status. The lack of effect of phytase on performance indicates that the birds did not suffer a deficiency of P. Under the circumstances of this trial, the P released by the phytase in the ileum was likely absorbed, but much of it would likely be excreted in the urine if there was not sufficient Ca with which to combine for bone deposition. Thus, there may have been an increase in the soluble P in excreta of such birds (Selle and Ravindran, 2007). Phytate, a myo-inositol ring with 6 phosphate groups attached, has enormous capacity to bind cations (such as Ca, P, Cu, and Zn) and form insoluble mineral-phytate complexes along with complexing with other nutrients, such as starch and amino acids, rendering them less digestible (Sebastian et al., 1998; Selle and Ravindran, 2007). The ability of poultry to hydrolyze the aforementioned complexes and release the bound nutrients depends on bird age, feed ingredients used, level of other nutrients, such as Ca and P, and also level of exogenous phytase supplementation (Rousseau et al., 2012; Liu et al., 2013; Walk et al., 2014). Exogenous phytase efficacy also depends upon its application dosage and dietary Ca level. In fact, keeping dietary Ca levels high, while maintaining dietary non-phytate P at a minimum level is the most effective way of compromising performance to such an extent that the benefits of added microbial phytase is maximized (Applegate et al., 2003; Plumstead et al., 2008). It has been postulated that reducing dietary Ca level significantly reduces the possibility of formation of insoluble-phytate complexes and thus more phytate is hydrolyzed in the intestinal tract by endogenous phytase (Applegate et al. 2003). This reduces the scope for an exogenous phytase response. On the other hand, phytase efficacy is highly dependent on dietary nPP level, with little effect noted in high P diets as was the case in the current trial. Therefore, since the basal diet had sufficient P to support the birds’ requirements, the lack of a significant effect of phytase in the present experiment is expected. However, the effects of phytase on reducing ileum ash and P contents indicates the functionality of the phytase even though a significant proportion of the absorbed P was probably excreted. In conclusion, our findings indicates that using a dietary Ca source such as CSM instead of CC allows use of a lower Ca diet with few consequences. When phytase is used in such diets, even at very high dosages, few benefits were observed, likely as a result of an imbalance between Ca and P, which was possibly made worse by phytase addition. Acknowledgements We gratefully acknowledge AB-Vista Feed Ingredients, (Marlborough, Wiltshire, SN8 4AN, United Kingdom) for providing phytase and CSM samples. REFERENCES AOAC. 1990. Official Methods of Analysis . 15th ed. Association of Official Analytical Chemists, Arlington, Virginia, USA. Applegate T. J., Angel R., Classen H. L.. 2003. 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Poultry ScienceOxford University Press

Published: Apr 1, 2018

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