animal
Animal (2014), 8:7, pp 1080–1088 © The Animal Consortium 2014 doi:10.1017/S1751731114001049
Nutritional geometry of calcium and phosphorus nutrition in broiler chicks. The effect of different dietary calcium and phosphorus concentrations and ratios on nutrient digestibility S. J. Wilkinson1†, E. J. Bradbury1, P. C. Thomson2, M. R. Bedford3 and A. J. Cowieson1 1 Poultry Research Foundation, The University of Sydney, Faculty of Veterinary Science, 425 Werombi Road, Camden, NSW 2570, Australia; 2Faculty of Veterinary Science, The University of Sydney, Camden, NSW 2570, Australia; 3AB Vista Feed Ingredients, Marlborough, Wiltshire, SN84AN, UK
(Received 15 December 2013; Accepted 7 March 2014; First published online 19 May 2014)
A total of 600 Ross 308-day-old male broiler chicks were used in a 28 day digestibility study to investigate the interaction between dietary calcium (Ca) and non-phytate phosphorus (nPP) on the digestibility of minerals and amino acids. Diets were formulated to be nutritionally adequate except for Ca and nPP. Fifteen mash diets based on corn and soya bean meal with varying concentrations of Ca (6.4 to 12.0 g/kg) and nPP (2.4 to 7.0 g/kg) were used. Diets were clustered around total densities of Ca and nPP of 12, 13.5 or 15.0 (g/kg) and within each density, a range of five Ca : nPP ratios (1.14 : 1, 1.5 : 1, 2.0 : 1, 2.75 : 1 and 4.0 : 1) were fed. Birds had free access to feed and water throughout the study. At day 28, birds were euthanised for the determination of apparent ileal mineral and amino acid digestibility. Data were modelled in R version 2.15 using a linear mixed-effects model and interrogation of the data was performed by fitting a low order polynomial function. At high Ca concentrations, increasing nPP led to an increase in the apparent digestibility of minerals. Apparent ileal digestibility of phosphorus (P) was enhanced with increasing dietary nPP up to 5.5 g/kg beyond which no improvements were found. Maximal Ca digestibility was found in diets with > 8.0 g/kg Ca with concomitant low concentrations of nPP. Diets with a broader Ca : nPP ratio improved the digestibility of Ca but were deleterious to the digestibility of P. In this study, apparent digestibility of amino acids was broadly unaffected by dietary Ca and nPP concentrations. However, interactions between Ca and nPP were observed for the digestibility of glutamine, tyrosine and methionine (all P < 0.001). Nitrogen digestibility showed discrete optima around 10.0 and 5.0 g/kg nPP and Na digestibility was maximised around 8 to 9.0 g/kg Ca and 4.5 to 5.4 g/kg nPP. These data show that the ratio of Ca : nPP is more influential to mineral digestibility than the absolute dietary concentration of each macro mineral. Keywords: broilers, calcium, geometric framework, phosphorus
Implications
Introduction
Calcium (Ca) and phosphorus (P) interactions on nutrient digestibility in broilers fed diets differing in concentration of these minerals were investigated using a geometric experimental approach. Generally, high concentrations of dietary Ca and low nPP, and high nPP and low Ca concentrations had negative effects on mineral digestibility. Amino acid digestibility was less influenced by dietary Ca and nPP. The data shows that the absolute amount of these minerals was less important than their relative proportions. Data from this study will contribute to a better understanding of Ca and P nutrition in broilers and the formulation of poultry diets.
Calcium (Ca) and phosphorous (P) requirements of poultry have been investigated extensively over the past 75 years (Driver et al., 2005). Despite decades of research investigating dietary concentrations of Ca and nPP required for optimum broiler performance, there is still no general agreement on what the appropriate concentrations of these two minerals and/or their relative proportions should be. Though Ca and P are interrelated in many biological functions, the requirement of these minerals is interdependent (Mello et al., 2012). Commercially, broilers are rarely, if ever, fed Ca concentrations recommended by the NRC (1994) and actual Ca intake is most probably 20% below these recommendations (Driver et al., 2005). The amount of Ca and nPP in poultry diets has both nutritional and economic implications. Excess dietary
†
E-mail: [email protected]
1080
Calcium phosphorus nutritional interactions Ca impedes the availability of other minerals such as P, magnesium, manganese and zinc and may also reduce the energy value of the diet (Driver et al., 2005; Selle et al., 2009). Decreasing dietary Ca concentrations may improve bird performance, P and nutrient digestibility; however, this may also lead to increased leg health problems (Wilkinson et al., 2011). The majority of published poultry nutritional research is based on altering a single food property at a time or changing multiple factors at once. However, the post-ingestive interactions of Ca and P in the digestive tract of poultry are highly complex and often difficult to interpret based on tabulated data. To better understand complex interactions such as those of Ca and P in poultry nutrition, it is necessary to use models that can assess several factors at once. The geometric framework graphically models outcomes in a geometric space that is fashioned by two or more axes that represent either nutritional, environmental or other variables that contribute to the fitness of the animal (Simpson and Raubenheimer, 2011). By graphing data as contour plots, conclusions may be visually assessed and more easily interpreted when compared with tabulated results. The objectives of the current study were to feed broilers diets varying in the concentrations and ratios of Ca and non-phytate phosphorus (nPP) to investigate their influence on nutrient digestibility. It was hypothesised that birds may increase digestibility of nutrients that are supplied insufficiently and decrease digestibility of nutrients supplied in excess. Material and methods All experimental procedures conducted in this study were in accordance with the University of Sydney Animal Ethics Committee and with the Australian code for the care and use of animals for scientific purposes (National Health and Medical Research Council, 2004).
Animals and housing Birds in this study were housed according to the conditions outlined by Bradbury et al. (2014). Briefly, a total of 600-dayold male Ross 308 chicks were obtained from a commercial hatchery. Birds were housed in metabolism cages within an environmentally controlled room maintained at 31°C for the first 5 days that was reduced by 0.5°C per day until 24°C (day 21). Photoperiod during the study was 23 L : 1 D (h) for the first 5 days and 18 L: 6 D (h) thereafter. Birds were fed a commercial broiler starter crumble diet for the first 7 days which provided 11.8 MJ/kg AME, 228 g/kg CP, 8.8 g/kg Ca and 4.4 g/kg nPP (Vella Stock Feeds, Plumpton, NSW, Australia). On day 7, all birds were weighed, wing tagged and randomly allocated to one of 15 dietary treatments. Each treatment was replicated five times with eight birds per replicate cage. Treatment diets were fed as a mash based on corn and soybean meal and were formulated to be nutritionally adequate (NRC, 1994) with the exception of Ca and nPP (Table 1). Diets were clustered around three total Ca + nPP densities of 12.0, 13.5 and 15.0 g/kg with five varying ratios
of total Ca : nPP (4 : 1, 2.75 : 1, 2 : 1 and 1.14 : 1) within each density. To calculate the apparent ileal digestibility (AID) coefficients for CP, amino acids, digestible energy (DE) and minerals, an indigestible marker (acid insoluble ash (AIA)) (Celite 281; Filchem Australia Pty Ltd, Castle Hill, NSW, Australia) was added to diets at a concentration of 20 g/kg. All birds were fed ad libitum and water was freely available. On day 28, birds were euthanised via an intravenous injection of sodium pentobarbitone (Lethabarb; Virbac Australia Pty Ltd, Milperra, NSW Australia). The contents of the lower ileum were collected according to the methods of Ravindran et al. (2005), pooled for each cage and immediately frozen and stored at − 20°C until further analysis.
Chemical analyses The ileal digesta samples were lyophilised and all samples were ground (Retsch ZM 100; Retsch GmbH, Haan, Germany) to pass through a 0.5 mm screen before chemical analyses. The gross energy (GE) and nitrogen (N) contents of diets and ileal digesta were determined. The diets and digesta were also analysed for minerals (P, Ca, Cu, K, Mg, Mn, Na, Sr, Fe and Zn). The GE of diets and excreta were determined using a Parr 1281 adiabatic bomb calorimeter (Parr Instrument Company, Moline, IL, USA) that was standardised with benzoic acid. Nitrogen concentration of samples was determined by the Dumas method using a FP-428 nitrogen analyser (LECO® Corporation, St. Joseph, MI, USA) as described by Sweeney (1989). Samples were wet acid digested using nitric acid and hydrogen peroxide (Peters et al., 2003) before the determination of mineral concentration by Inductively Coupled Plasma-Optical Emission Spectroscopy using a Perkin Elmer OPTIMA 7300 (Perkin Elmer Inc., Waltham, MA, USA). Amino acid concentrations in the diet and dried ileal digesta samples were determined using a Waters AccQTag Ultra amino acid analysis system following acid hydrolysis in 6 M HCl at 110°C. The AIA component of dried diets and ileal digesta samples were determined according to the method of Siriwan et al. (1993). The AID coefficient of N, DE, amino acids and minerals were calculated as per Ravindran et al. (2001). Statistical analysis All data were analysed using the linear mixed-effects model in R version 2.15 (R Development Core Team, 2011). Fixed effects were dietary Ca and nPP and block was included as a random effect. Treatments are represented as dots overlaid on the contour plots. Interrogation of the data was performed using a quadratic polynomial function and the terms Ca and P presented on the contour plots are composites of linear and quadratic terms. Differences were considered significant at P < 0.05. Results Bird performance, tibia ash and latency to lie results are presented in Bradbury et al. (2014). The analysed Ca and P concentrations (Table 1) were within acceptable ranges 1081
Treatment Ca (g/kg) nPP (g/kg) Ca : nPP (g : g) Corn Soya bean meal Sunflower oil Salt Sodium bicarbonate DL-Methionine Lysine HCl Threonine Limestone Dicalcium PO4 Celite1 Vitamin premix2 Zinc bacitracin3 Calculated AME (MJ/kg) CP (N × 6.25) Analysed GE (kcal) CP (N × 6.25) Ca Total P
1 12.0 3.0 4.00
2 11.0 4.0 2.75
3 10.0 5.0 2.00
4
5
9.0 6.0 1.50
8.0 7.0 1.14
6 10.8 2.7 4.0
7
8
9
9.9 3.6 2.75
9.0 4.5 2.0
8.1 5.4 1.50
10 7.2 6.3 1.14
11 9.6 2.4 4.00
12 8.8 3.2 2.75
13 8.0 4.0 2.00
14 7.2 4.8 1.50
15 6.4 5.6 1.14
665.2 270.4 18.3 1.3 4.0 2.8 3.6 0.8 22.5 11.1 20.0 2.0 0.3
666.3 270.2 17.9 1.3 4.0 2.8 3.6 0.8 16.5 16.6 20.0 2.0 0.3
667.4 270.0 17.6 1.3 4.0 2.8 3.6 0.8 10.5 22.0 20.0 2.0 0.3
668.5 269.8 17.2 1.3 4.0 2.8 3.6 0.8 4.4 27.5 20.0 2.0 0.3
666.4 270.2 17.9 1.3 4.0 2.8 3.6 0.8 0.0 33.0 20.0 2.0 0.3
672.8 269.1 15.8 1.3 4.0 2.8 3.6 0.8 20.4 9.4 20.0 2.0 0.3
673.8 268.9 15.5 1.3 4.0 2.8 3.6 0.8 15.0 14.3 20.0 2.0 0.3
674.8 268.7 15.2 1.3 4.0 2.8 3.6 0.8 9.5 19.3 20.0 2.0 0.3
675.7 268.5 14.9 1.3 4.0 2.8 3.6 0.8 4.1 24.2 20.0 2.0 0.3
674.1 268.8 15.4 1.3 4.0 2.8 3.6 0.8 0.0 29.1 20.0 2.0 0.3
680.4 267.7 13.4 1.3 4.1 2.8 3.6 0.8 18.2 7.8 20.0 2.0 0.3
679.6 267.9 13.6 1.3 4.0 2.8 3.6 0.8 12.1 14.3 20.0 2.0 0.3
682.1 267.4 12.8 1.3 4.1 2.8 3.6 0.8 8.6 16.5 20.0 2.0 0.3
683.0 267.2 12.5 1.3 4.1 2.8 3.6 0.8 3.8 20.9 20.0 2.0 0.3
681.8 267.5 12.9 1.3 4.0 2.8 3.6 0.8 0.0 25.3 20.0 2.0 0.3
12.98 191
12.98 191
12.98 191
12.98 191
12.98 191
12.98 191
12.98 191
12.98 191
12.98 191
12.98 191
12.98 191
12.98 191
12.98 191
12.98 191
12.98 191
3897 17.5 11.5 4.6
3893 17.8 11.3 5.4
3912 17.9 11.0 6.5
3852 17.8 10.1 7.0
3893 17.3 9.4 7.5
3871 17.2 12.7 4.6
3909 17.4 11.9 5.4
3912 17.7 12.2 6.4
3924 17.4 11.5 7.1
3907 17.4 12.0 7.9
3895 17.7 12.9 4.7
3940 17.5 10.7 5.4
3943 17.0 9.4 5.6
3883 17.0 9.9 6.3
3905 17.3 9.8 6.9
Ca = calcium; nPP = non-phytate phosphorus; P = phosphorus. 1 Filchem Australia Pty Ltd, Castle Hill, NSW, Australia. 2 Supplied per kg of diet: retinol, 3600 μg; cholecalciferol, 125 μg; α-tocopherol, 50 mg; menadione, 3 mg; thiamine, 3 mg; riboflavin, 9 mg; pyridoxine, 5 mg; cobalamin 25 μg; niacin, 50 mg; pantothenic acid, 18 mg; folic acid, 2 mg; biotin, 200 μg; Cu, 20 mg; Fe, 40 mg; Mn, 110 mg; Co, 250 μg; I, 1 mg; Mo, 2 mg; Zn, 90 mg; Se, 300 μg; ethoxyquin, 125 mg. 3 Albac, G 150 (Philbro Animal Health Pty Ltd, Girraween, NSW, Australia)
Wilkinson, Bradbury, Thomson, Bedford and Cowieson
1082
Table 1 Ingredient and nutrient specifications (as fed g/kg) of the experimental diets fed to Ross 308 broilers (day 7 to day 28)
< 0.042 < 0.062 0.085 < 0.001 0.359 0.567 < 0.001 0.746 < 0.001 < 0.001 0.004 0.001 < 0.001 < 0.001 < 0.001 0.005 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.039 < 0.001 0.03 < 0.001 < 0.001 < 0.001 < 0.001 0.007 0.036 < 0.001 < 0.001 0.55 0.084 0.201 < 0.001 < 0.001 < 0.001 < 0.001 Mean values from five treatment replicates. Pooled s.e.m. 2
1
DM DE N Ca P Mg Na K Fe Cu Mn Zn Sr
73.5 74.9 84.0 47.0 40.7 17.6 − 32.2 81.9 76.3 35.1 9.5 13.8 26.1
75.5 77.6 85.6 35.4 43.7 29.1 − 10.6 87.8 74.8 21.0 9.7 17.9 19.3
75.6 78.0 86.1 27.6 44.5 30.9 − 2.7 90.3 73.2 11.2 8.0 18.4 13.0
73.8 76.2 85.4 23.6 43.2 23.3 − 8.4 89.3 71.8 5.7 4.4 15.2 7.2
70.0 72.0 83.7 23.5 39.7 6.1 − 27.8 84.9 70.4 4.4 − 1.0 8.5 1.9
68.4 69.9 82.1 54.4 31.1 − 2.0 − 19.2 78.9 81.3 33.7 3.8 11.0 27.2
71.6 73.8 84.3 42.4 35.7 13.0 5.5 85.1 81.1 23.1 6.6 16.7 22.0
73.2 75.8 85.5 33.6 38.6 20.2 19.2 88.5 81.0 16.0 7.8 19.4 17.3
73.3 76.0 85.8 27.9 39.7 19.7 21.8 89.1 80.8 12.4 7.6 19.3 13.0
71.8 74.4 85.3 25.3 39.0 11.5 13.4 86.9 80.7 12.3 5.8 16.2 9.1
61.3 62.8 76.4 50.2 19.9 − 23.6 − 61.2 75.8 74.1 8.4 − 14.7 − 7.5 13.7
68.3 70.7 78.9 36.9 25.7 − 6.1 − 34.5 82.1 78.1 0.9 − 9.9 − 0.7 10.0
68.2 70.9 80.7 28.4 30.0 5.3 − 16.4 86.2 76.8 − 3.9 − 6.3 3.9 6.6
69.8 72.7 81.9 21.9 33.0 10.5 − 7.1 88.1 76.9 − 5.9 − 3.9 6.1 3.6
70.2 73.1 82.3 17.3 34.7 9.7 − 6.5 87.8 77.8 − 5.1 − 2.7 6.0 0.9
0.7 0.8 1.3 2.6 1.7 2.7 6.1 0.7 1.2 4.1 2.8 2.7 2.4
nPP Ca s.e.m.2 6.4 5.6 7.2 4.8 8.0 4.0 8.8 3.2 9.6 2.4 7.2 6.3 8.1 5.4 9.0 4.5 9.9 3.6 10.8 2.7 8.0 7.0 9.0 6.0 10.0 5.0 11.0 4.0 12.0 3.0
Table 2 Interaction of dietary Ca and nPP concentration (g/kg) on the apparent ileal digestibility (%) of dry matter (DM), digestible energy (DE), nitrogen (N) and minerals in d 28 broilers1
when mixing and assay errors were considered. The effect of dietary Ca and nPP on the AID of N, DM, DE and minerals are presented in Table 2. The AID of DM and DE were highest in birds fed diets containing 4 to 5 g/kg nPP and 10 to 11 g/kg Ca. Wide Ca : nPP or medium Ca : nPP ratios reduced the AID of DM and DE within each Ca : nPP cluster resulting in a significant interaction between dietary Ca and nPP. Both total Ca and nPP concentrations influenced the digestibility of N (P < 0.01) and was greatest in birds fed diets containing ~ 8 to 10 g/kg Ca and 4.0 to 6.0 g/kg nPP. The AID of Na increased within each Ca : nPP cluster as the ratio of Ca : nPP narrowed and conversely as this ratio widened the AID of Na was decreased leading to a significant interaction between the two macro minerals (P < 0.001). Maximal Na digestibility found in birds fed 8.1 g/kg Ca and 5.4 g/kg nPP. Diets containing high Ca (>8.0 g/kg) and low nPP (9.0 g/kg) and low nPP (10 g/kg Ca and < 3.5 g/kg nPP and had significant interactions between Ca and nPP (Table 2). Generally, increased dietary Ca concentrations improved the ileal digestibility of P at lower dietary P concentrations and less so for higher dietary P concentrations (Figure 4). Phosphorus and Mg digestibility were greatest in birds fed 10.0 g/kg Ca and 5.0 g/kg nPP. The ileal digestibility of K in high Ca (>10g/kg) diets was substantially improved with increased nPP, however, this improvement abated above 5.0 g/kg nPP. Ileal digestibility of Fe did not resemble any of the surface plots of the other minerals (Figure 5). An interaction between dietary Ca and nPP was reported (P
Animal (2014), 8:7, pp 1080–1088 © The Animal Consortium 2014 doi:10.1017/S1751731114001049
Nutritional geometry of calcium and phosphorus nutrition in broiler chicks. The effect of different dietary calcium and phosphorus concentrations and ratios on nutrient digestibility S. J. Wilkinson1†, E. J. Bradbury1, P. C. Thomson2, M. R. Bedford3 and A. J. Cowieson1 1 Poultry Research Foundation, The University of Sydney, Faculty of Veterinary Science, 425 Werombi Road, Camden, NSW 2570, Australia; 2Faculty of Veterinary Science, The University of Sydney, Camden, NSW 2570, Australia; 3AB Vista Feed Ingredients, Marlborough, Wiltshire, SN84AN, UK
(Received 15 December 2013; Accepted 7 March 2014; First published online 19 May 2014)
A total of 600 Ross 308-day-old male broiler chicks were used in a 28 day digestibility study to investigate the interaction between dietary calcium (Ca) and non-phytate phosphorus (nPP) on the digestibility of minerals and amino acids. Diets were formulated to be nutritionally adequate except for Ca and nPP. Fifteen mash diets based on corn and soya bean meal with varying concentrations of Ca (6.4 to 12.0 g/kg) and nPP (2.4 to 7.0 g/kg) were used. Diets were clustered around total densities of Ca and nPP of 12, 13.5 or 15.0 (g/kg) and within each density, a range of five Ca : nPP ratios (1.14 : 1, 1.5 : 1, 2.0 : 1, 2.75 : 1 and 4.0 : 1) were fed. Birds had free access to feed and water throughout the study. At day 28, birds were euthanised for the determination of apparent ileal mineral and amino acid digestibility. Data were modelled in R version 2.15 using a linear mixed-effects model and interrogation of the data was performed by fitting a low order polynomial function. At high Ca concentrations, increasing nPP led to an increase in the apparent digestibility of minerals. Apparent ileal digestibility of phosphorus (P) was enhanced with increasing dietary nPP up to 5.5 g/kg beyond which no improvements were found. Maximal Ca digestibility was found in diets with > 8.0 g/kg Ca with concomitant low concentrations of nPP. Diets with a broader Ca : nPP ratio improved the digestibility of Ca but were deleterious to the digestibility of P. In this study, apparent digestibility of amino acids was broadly unaffected by dietary Ca and nPP concentrations. However, interactions between Ca and nPP were observed for the digestibility of glutamine, tyrosine and methionine (all P < 0.001). Nitrogen digestibility showed discrete optima around 10.0 and 5.0 g/kg nPP and Na digestibility was maximised around 8 to 9.0 g/kg Ca and 4.5 to 5.4 g/kg nPP. These data show that the ratio of Ca : nPP is more influential to mineral digestibility than the absolute dietary concentration of each macro mineral. Keywords: broilers, calcium, geometric framework, phosphorus
Implications
Introduction
Calcium (Ca) and phosphorus (P) interactions on nutrient digestibility in broilers fed diets differing in concentration of these minerals were investigated using a geometric experimental approach. Generally, high concentrations of dietary Ca and low nPP, and high nPP and low Ca concentrations had negative effects on mineral digestibility. Amino acid digestibility was less influenced by dietary Ca and nPP. The data shows that the absolute amount of these minerals was less important than their relative proportions. Data from this study will contribute to a better understanding of Ca and P nutrition in broilers and the formulation of poultry diets.
Calcium (Ca) and phosphorous (P) requirements of poultry have been investigated extensively over the past 75 years (Driver et al., 2005). Despite decades of research investigating dietary concentrations of Ca and nPP required for optimum broiler performance, there is still no general agreement on what the appropriate concentrations of these two minerals and/or their relative proportions should be. Though Ca and P are interrelated in many biological functions, the requirement of these minerals is interdependent (Mello et al., 2012). Commercially, broilers are rarely, if ever, fed Ca concentrations recommended by the NRC (1994) and actual Ca intake is most probably 20% below these recommendations (Driver et al., 2005). The amount of Ca and nPP in poultry diets has both nutritional and economic implications. Excess dietary
†
E-mail: [email protected]
1080
Calcium phosphorus nutritional interactions Ca impedes the availability of other minerals such as P, magnesium, manganese and zinc and may also reduce the energy value of the diet (Driver et al., 2005; Selle et al., 2009). Decreasing dietary Ca concentrations may improve bird performance, P and nutrient digestibility; however, this may also lead to increased leg health problems (Wilkinson et al., 2011). The majority of published poultry nutritional research is based on altering a single food property at a time or changing multiple factors at once. However, the post-ingestive interactions of Ca and P in the digestive tract of poultry are highly complex and often difficult to interpret based on tabulated data. To better understand complex interactions such as those of Ca and P in poultry nutrition, it is necessary to use models that can assess several factors at once. The geometric framework graphically models outcomes in a geometric space that is fashioned by two or more axes that represent either nutritional, environmental or other variables that contribute to the fitness of the animal (Simpson and Raubenheimer, 2011). By graphing data as contour plots, conclusions may be visually assessed and more easily interpreted when compared with tabulated results. The objectives of the current study were to feed broilers diets varying in the concentrations and ratios of Ca and non-phytate phosphorus (nPP) to investigate their influence on nutrient digestibility. It was hypothesised that birds may increase digestibility of nutrients that are supplied insufficiently and decrease digestibility of nutrients supplied in excess. Material and methods All experimental procedures conducted in this study were in accordance with the University of Sydney Animal Ethics Committee and with the Australian code for the care and use of animals for scientific purposes (National Health and Medical Research Council, 2004).
Animals and housing Birds in this study were housed according to the conditions outlined by Bradbury et al. (2014). Briefly, a total of 600-dayold male Ross 308 chicks were obtained from a commercial hatchery. Birds were housed in metabolism cages within an environmentally controlled room maintained at 31°C for the first 5 days that was reduced by 0.5°C per day until 24°C (day 21). Photoperiod during the study was 23 L : 1 D (h) for the first 5 days and 18 L: 6 D (h) thereafter. Birds were fed a commercial broiler starter crumble diet for the first 7 days which provided 11.8 MJ/kg AME, 228 g/kg CP, 8.8 g/kg Ca and 4.4 g/kg nPP (Vella Stock Feeds, Plumpton, NSW, Australia). On day 7, all birds were weighed, wing tagged and randomly allocated to one of 15 dietary treatments. Each treatment was replicated five times with eight birds per replicate cage. Treatment diets were fed as a mash based on corn and soybean meal and were formulated to be nutritionally adequate (NRC, 1994) with the exception of Ca and nPP (Table 1). Diets were clustered around three total Ca + nPP densities of 12.0, 13.5 and 15.0 g/kg with five varying ratios
of total Ca : nPP (4 : 1, 2.75 : 1, 2 : 1 and 1.14 : 1) within each density. To calculate the apparent ileal digestibility (AID) coefficients for CP, amino acids, digestible energy (DE) and minerals, an indigestible marker (acid insoluble ash (AIA)) (Celite 281; Filchem Australia Pty Ltd, Castle Hill, NSW, Australia) was added to diets at a concentration of 20 g/kg. All birds were fed ad libitum and water was freely available. On day 28, birds were euthanised via an intravenous injection of sodium pentobarbitone (Lethabarb; Virbac Australia Pty Ltd, Milperra, NSW Australia). The contents of the lower ileum were collected according to the methods of Ravindran et al. (2005), pooled for each cage and immediately frozen and stored at − 20°C until further analysis.
Chemical analyses The ileal digesta samples were lyophilised and all samples were ground (Retsch ZM 100; Retsch GmbH, Haan, Germany) to pass through a 0.5 mm screen before chemical analyses. The gross energy (GE) and nitrogen (N) contents of diets and ileal digesta were determined. The diets and digesta were also analysed for minerals (P, Ca, Cu, K, Mg, Mn, Na, Sr, Fe and Zn). The GE of diets and excreta were determined using a Parr 1281 adiabatic bomb calorimeter (Parr Instrument Company, Moline, IL, USA) that was standardised with benzoic acid. Nitrogen concentration of samples was determined by the Dumas method using a FP-428 nitrogen analyser (LECO® Corporation, St. Joseph, MI, USA) as described by Sweeney (1989). Samples were wet acid digested using nitric acid and hydrogen peroxide (Peters et al., 2003) before the determination of mineral concentration by Inductively Coupled Plasma-Optical Emission Spectroscopy using a Perkin Elmer OPTIMA 7300 (Perkin Elmer Inc., Waltham, MA, USA). Amino acid concentrations in the diet and dried ileal digesta samples were determined using a Waters AccQTag Ultra amino acid analysis system following acid hydrolysis in 6 M HCl at 110°C. The AIA component of dried diets and ileal digesta samples were determined according to the method of Siriwan et al. (1993). The AID coefficient of N, DE, amino acids and minerals were calculated as per Ravindran et al. (2001). Statistical analysis All data were analysed using the linear mixed-effects model in R version 2.15 (R Development Core Team, 2011). Fixed effects were dietary Ca and nPP and block was included as a random effect. Treatments are represented as dots overlaid on the contour plots. Interrogation of the data was performed using a quadratic polynomial function and the terms Ca and P presented on the contour plots are composites of linear and quadratic terms. Differences were considered significant at P < 0.05. Results Bird performance, tibia ash and latency to lie results are presented in Bradbury et al. (2014). The analysed Ca and P concentrations (Table 1) were within acceptable ranges 1081
Treatment Ca (g/kg) nPP (g/kg) Ca : nPP (g : g) Corn Soya bean meal Sunflower oil Salt Sodium bicarbonate DL-Methionine Lysine HCl Threonine Limestone Dicalcium PO4 Celite1 Vitamin premix2 Zinc bacitracin3 Calculated AME (MJ/kg) CP (N × 6.25) Analysed GE (kcal) CP (N × 6.25) Ca Total P
1 12.0 3.0 4.00
2 11.0 4.0 2.75
3 10.0 5.0 2.00
4
5
9.0 6.0 1.50
8.0 7.0 1.14
6 10.8 2.7 4.0
7
8
9
9.9 3.6 2.75
9.0 4.5 2.0
8.1 5.4 1.50
10 7.2 6.3 1.14
11 9.6 2.4 4.00
12 8.8 3.2 2.75
13 8.0 4.0 2.00
14 7.2 4.8 1.50
15 6.4 5.6 1.14
665.2 270.4 18.3 1.3 4.0 2.8 3.6 0.8 22.5 11.1 20.0 2.0 0.3
666.3 270.2 17.9 1.3 4.0 2.8 3.6 0.8 16.5 16.6 20.0 2.0 0.3
667.4 270.0 17.6 1.3 4.0 2.8 3.6 0.8 10.5 22.0 20.0 2.0 0.3
668.5 269.8 17.2 1.3 4.0 2.8 3.6 0.8 4.4 27.5 20.0 2.0 0.3
666.4 270.2 17.9 1.3 4.0 2.8 3.6 0.8 0.0 33.0 20.0 2.0 0.3
672.8 269.1 15.8 1.3 4.0 2.8 3.6 0.8 20.4 9.4 20.0 2.0 0.3
673.8 268.9 15.5 1.3 4.0 2.8 3.6 0.8 15.0 14.3 20.0 2.0 0.3
674.8 268.7 15.2 1.3 4.0 2.8 3.6 0.8 9.5 19.3 20.0 2.0 0.3
675.7 268.5 14.9 1.3 4.0 2.8 3.6 0.8 4.1 24.2 20.0 2.0 0.3
674.1 268.8 15.4 1.3 4.0 2.8 3.6 0.8 0.0 29.1 20.0 2.0 0.3
680.4 267.7 13.4 1.3 4.1 2.8 3.6 0.8 18.2 7.8 20.0 2.0 0.3
679.6 267.9 13.6 1.3 4.0 2.8 3.6 0.8 12.1 14.3 20.0 2.0 0.3
682.1 267.4 12.8 1.3 4.1 2.8 3.6 0.8 8.6 16.5 20.0 2.0 0.3
683.0 267.2 12.5 1.3 4.1 2.8 3.6 0.8 3.8 20.9 20.0 2.0 0.3
681.8 267.5 12.9 1.3 4.0 2.8 3.6 0.8 0.0 25.3 20.0 2.0 0.3
12.98 191
12.98 191
12.98 191
12.98 191
12.98 191
12.98 191
12.98 191
12.98 191
12.98 191
12.98 191
12.98 191
12.98 191
12.98 191
12.98 191
12.98 191
3897 17.5 11.5 4.6
3893 17.8 11.3 5.4
3912 17.9 11.0 6.5
3852 17.8 10.1 7.0
3893 17.3 9.4 7.5
3871 17.2 12.7 4.6
3909 17.4 11.9 5.4
3912 17.7 12.2 6.4
3924 17.4 11.5 7.1
3907 17.4 12.0 7.9
3895 17.7 12.9 4.7
3940 17.5 10.7 5.4
3943 17.0 9.4 5.6
3883 17.0 9.9 6.3
3905 17.3 9.8 6.9
Ca = calcium; nPP = non-phytate phosphorus; P = phosphorus. 1 Filchem Australia Pty Ltd, Castle Hill, NSW, Australia. 2 Supplied per kg of diet: retinol, 3600 μg; cholecalciferol, 125 μg; α-tocopherol, 50 mg; menadione, 3 mg; thiamine, 3 mg; riboflavin, 9 mg; pyridoxine, 5 mg; cobalamin 25 μg; niacin, 50 mg; pantothenic acid, 18 mg; folic acid, 2 mg; biotin, 200 μg; Cu, 20 mg; Fe, 40 mg; Mn, 110 mg; Co, 250 μg; I, 1 mg; Mo, 2 mg; Zn, 90 mg; Se, 300 μg; ethoxyquin, 125 mg. 3 Albac, G 150 (Philbro Animal Health Pty Ltd, Girraween, NSW, Australia)
Wilkinson, Bradbury, Thomson, Bedford and Cowieson
1082
Table 1 Ingredient and nutrient specifications (as fed g/kg) of the experimental diets fed to Ross 308 broilers (day 7 to day 28)
< 0.042 < 0.062 0.085 < 0.001 0.359 0.567 < 0.001 0.746 < 0.001 < 0.001 0.004 0.001 < 0.001 < 0.001 < 0.001 0.005 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.039 < 0.001 0.03 < 0.001 < 0.001 < 0.001 < 0.001 0.007 0.036 < 0.001 < 0.001 0.55 0.084 0.201 < 0.001 < 0.001 < 0.001 < 0.001 Mean values from five treatment replicates. Pooled s.e.m. 2
1
DM DE N Ca P Mg Na K Fe Cu Mn Zn Sr
73.5 74.9 84.0 47.0 40.7 17.6 − 32.2 81.9 76.3 35.1 9.5 13.8 26.1
75.5 77.6 85.6 35.4 43.7 29.1 − 10.6 87.8 74.8 21.0 9.7 17.9 19.3
75.6 78.0 86.1 27.6 44.5 30.9 − 2.7 90.3 73.2 11.2 8.0 18.4 13.0
73.8 76.2 85.4 23.6 43.2 23.3 − 8.4 89.3 71.8 5.7 4.4 15.2 7.2
70.0 72.0 83.7 23.5 39.7 6.1 − 27.8 84.9 70.4 4.4 − 1.0 8.5 1.9
68.4 69.9 82.1 54.4 31.1 − 2.0 − 19.2 78.9 81.3 33.7 3.8 11.0 27.2
71.6 73.8 84.3 42.4 35.7 13.0 5.5 85.1 81.1 23.1 6.6 16.7 22.0
73.2 75.8 85.5 33.6 38.6 20.2 19.2 88.5 81.0 16.0 7.8 19.4 17.3
73.3 76.0 85.8 27.9 39.7 19.7 21.8 89.1 80.8 12.4 7.6 19.3 13.0
71.8 74.4 85.3 25.3 39.0 11.5 13.4 86.9 80.7 12.3 5.8 16.2 9.1
61.3 62.8 76.4 50.2 19.9 − 23.6 − 61.2 75.8 74.1 8.4 − 14.7 − 7.5 13.7
68.3 70.7 78.9 36.9 25.7 − 6.1 − 34.5 82.1 78.1 0.9 − 9.9 − 0.7 10.0
68.2 70.9 80.7 28.4 30.0 5.3 − 16.4 86.2 76.8 − 3.9 − 6.3 3.9 6.6
69.8 72.7 81.9 21.9 33.0 10.5 − 7.1 88.1 76.9 − 5.9 − 3.9 6.1 3.6
70.2 73.1 82.3 17.3 34.7 9.7 − 6.5 87.8 77.8 − 5.1 − 2.7 6.0 0.9
0.7 0.8 1.3 2.6 1.7 2.7 6.1 0.7 1.2 4.1 2.8 2.7 2.4
nPP Ca s.e.m.2 6.4 5.6 7.2 4.8 8.0 4.0 8.8 3.2 9.6 2.4 7.2 6.3 8.1 5.4 9.0 4.5 9.9 3.6 10.8 2.7 8.0 7.0 9.0 6.0 10.0 5.0 11.0 4.0 12.0 3.0
Table 2 Interaction of dietary Ca and nPP concentration (g/kg) on the apparent ileal digestibility (%) of dry matter (DM), digestible energy (DE), nitrogen (N) and minerals in d 28 broilers1
when mixing and assay errors were considered. The effect of dietary Ca and nPP on the AID of N, DM, DE and minerals are presented in Table 2. The AID of DM and DE were highest in birds fed diets containing 4 to 5 g/kg nPP and 10 to 11 g/kg Ca. Wide Ca : nPP or medium Ca : nPP ratios reduced the AID of DM and DE within each Ca : nPP cluster resulting in a significant interaction between dietary Ca and nPP. Both total Ca and nPP concentrations influenced the digestibility of N (P < 0.01) and was greatest in birds fed diets containing ~ 8 to 10 g/kg Ca and 4.0 to 6.0 g/kg nPP. The AID of Na increased within each Ca : nPP cluster as the ratio of Ca : nPP narrowed and conversely as this ratio widened the AID of Na was decreased leading to a significant interaction between the two macro minerals (P < 0.001). Maximal Na digestibility found in birds fed 8.1 g/kg Ca and 5.4 g/kg nPP. Diets containing high Ca (>8.0 g/kg) and low nPP (9.0 g/kg) and low nPP (10 g/kg Ca and < 3.5 g/kg nPP and had significant interactions between Ca and nPP (Table 2). Generally, increased dietary Ca concentrations improved the ileal digestibility of P at lower dietary P concentrations and less so for higher dietary P concentrations (Figure 4). Phosphorus and Mg digestibility were greatest in birds fed 10.0 g/kg Ca and 5.0 g/kg nPP. The ileal digestibility of K in high Ca (>10g/kg) diets was substantially improved with increased nPP, however, this improvement abated above 5.0 g/kg nPP. Ileal digestibility of Fe did not resemble any of the surface plots of the other minerals (Figure 5). An interaction between dietary Ca and nPP was reported (P
