Variability of prececal phosphorus digestibility of triticale and wheat in broiler chickens

Variability of prececal phosphorus digestibility of triticale and wheat in broiler chickens Abstract The aim of this study was to evaluate the P digestibility of different wheat and triticale genotypes in growing broiler chickens. Additionally, the relationships between physical or chemical properties of genotypes and P digestibility were determined. A low P, low Ca basal diet based on cornstarch and soybean meal was supplemented with 20% or 40% of 8 different wheat or triticale genotypes at the expense of cornstarch. Experimental diets were fed to broilers between d 20 and 26 of age before digesta samples were collected from their lower ileum for determination of prececal P digestibility (pcdP). Triticale-based diets had an average pcdP of 54%. Neither the concentration of triticale nor the genotype itself affected the pcdP of diets. The pcdP of triticale genotypes calculated by linear regression analysis ranged from 53% to 78%. No correlations were found between physical or chemical properties (viscosity, phytase activity, total and phytate P contents) and the pcdP of triticale genotypes. In contrast, a significant (P < 0.05) effect of genotype and wheat concentration was observed in wheat-based diets. The average pcdP of diets decreased from 60% to 50% by increasing the wheat concentration from 20% to 40%. As no linear relationship was observed between P intake and the amount of pcdP in the diet, the pcdP (%) of wheat genotypes was calculated separately for both concentrations, and accounted for 38% to 67% and 20% to 38% with 20% and 40% wheat inclusion in diets, respectively. Single chemical and physical characteristics could not explain the observed differences in P digestibility. Our results revealed a high variation in the pcdP of different wheat and triticale genotypes that should be considered in diet formulation. However, further research is needed to identify factors that contribute to variation of P digestibility in both grains. INTRODUCTION Phosphorus (P) is an element with special relevance for sustainable poultry feeding. In non-ruminants, the digestibility of plant P bound to phytate, a salt of phytic acid that represents the main storage form of plant P, is limited due to the lack of sufficient production of endogenous phytase in the upper gastrointestinal tract. Thus, diets are usually supplemented with highly digestible feed phosphates or phytase to meet the animal's P requirement. However, these supplements are expensive and global resources of raw phosphates are limited (Abelson, 1999). Moreover, excessive P intake can be detrimental to the environment in regions of high livestock density (Sharpley, 1999; Rodehutscord, 2008). Thus, for a responsible handling of P sources and a precise supply of dietary P to animals a sound knowledge on different feed raw materials, which differ considerably in their P digestibility, is required (World's Poultry Science Association [WPSA], 2013). Therefore, the WPSA recently published a standard protocol for testing the P digestibility of feed raw materials (WPSA, 2013). Wheat is an important grain in poultry nutrition (Coskuntuna et al., 2008; Stef et al., 2013) and is generally included at up to 55% in broiler diets (Gutierrez et al., 2009). Among cereals, wheat contains moderate total P (∼3.7 g/kg dry matter [DM]) and phytate P (∼1.9 g/kg DM) concentrations, while its intrinsic phytase activity is high (∼1,850 units/kg DM) (Rodehutscord et al., 2016). Thus, wheat can contribute considerably to an animal's P supply. However, in the only studies reported to date that have investigated wheat using the WPSA (2013) protocol, the prececal P digestibility (pcdP) of wheat was 18% and 46% in 4-5 wk-old broilers (Mutucumarana et al., 2014a; Kupcikova et al., 2017). Further reports in the literature refer only to the pcdP or P retention from wheat-based complete diets (van der Klis et al., 1995; Rutherfurd et al., 2002; Wu et al., 2004; Afsharmanesh et al., 2008). Afsharmanesh et al. (2008) used 3-wk-old broilers and fed diets based on 80% Durum or hard red spring wheat and found pcdP values of 60% and 64%, respectively, thus indicating a significant effect of the wheat variety on the pcdP. Different wheat varieties may differ in contents of total P, phytate P, soluble and insoluble non-starch polysaccharides (NSP) and their intrinsic phytase activity and extract viscosity (van der Klis et al., 1995; Zyla et al., 1999; Erdal et al., 2002; Steiner et al., 2007; Rodehutscord et al., 2016) which may affect the pcdP in vivo (van der Klis et al., 1995) and the release of inorganic P in vitro (Zyla et al., 1999). Triticale, an intergenic hybrid, is an alternative cereal grain combining the feeding characteristics of wheat and winter hardiness, drought, and disease resistance of rye (Boros, 1999). Thus, triticale is more important in regions where wheat or corn cannot be grown (Djekic et al., 2011). Modern high-yielding varieties with improved nutritional properties have the potential to replace wheat and corn in poultry diets (Widodo et al., 2015). However, more research is necessary to evaluate the nutritive value of triticale, especially with respect to its P digestibility. On average, concentrations of phytate P, total and soluble NSP are similar to those found in wheat, while the total P content, intrinsic phytase activity, and extract viscosity are higher in triticale than in wheat. However, the chemical composition of different triticale varieties may vary considerably (Jondreville et al., 2007; Rodehutscord et al., 2016). Hence, the increasing use of triticale and the differences found between varieties necessitate an assessment of the different varieties’ P digestibility in poultry. The objective of the present study was to determine the pcdP of different wheat and triticale genotypes grown under similar environmental and agronomic conditions using the protocol recommended by the WPSA (2013). Moreover, the relationships between physical or chemical properties of grains and the pcdP of genotypes were investigated. MATERIALS AND METHODS This study was part of the collaborative research project referred to as GrainUp (www.grain-up.de). The 8 triticale and 8 wheat genotypes used in this study were grown under similar environmental and agronomic conditions, but specific for each species. Cultivation and processing, as well as physical and chemical properties of these genotypes are described in detail elsewhere (Rodehutscord et al., 2016). Genotypes used in this study were numbered in the same way as in Rodehutscord et al. (2016). The methodological approach used in this study followed the protocol recommended to determine the P digestibility in poultry from plant sources (WPSA, 2013). Experimental Diets Two cornstarch-potato protein-based basal diets (BD) were formulated to contain adequate concentrations of all nutrients according to the recommendations of the Gesellschaft für Ernährungsphysiologie (GfE, 1999), with the exceptions of Ca and P (Table 1). The 2 BD contained either 10% of a mixture of the 8 triticale genotypes (experiment 1) or 10% of a mixture of the 8 wheat genotypes (experiment 2) tested in this study. In 32 further experimental diets (genotype diets, GD), each of the 8 triticale and wheat genotypes was added to the BD at 2 different concentrations (20% or 40%, Table 1) at the expense of cornstarch, thus making the triticale or wheat genotype the only source of variation in P content of the diets. Prior to mixing, cereal grains were ground to pass through a 2-mm sieve screen. Limestone was also added to the GD at the expense of cornstarch to retain a constant Ca: P ratio in all diets. Titanium dioxide was included (0.5%) as an indigestible marker. Analyzed phytase activity and concentrations of total P and Ca of the experimental diets are presented in Table 2. Phytase activity ranged from <100 to 890 FTU/kg DM in triticale-based diets and from 300 to 1720 FTU/kg DM in wheat-based diets. The concentrations of total P in experimental diets with triticale ranged from 2.64 to 4.33 and from 2.5 to 4.36 g/kg DM in diets with wheat. The Ca: total P ratio in diets with triticale or wheat was on average (standard deviation [SD] in parentheses) 1.41:1.0 (0.06) and 1.33:1.0 (0.07), respectively, which was very close to the ratio (1.3:1.0 to 1.4:1.0) recommended in the WPSA protocol (WPSA, 2013). Analyzed titanium dioxide concentrations in diets ranged from 4.8 to 4.9 g/kg and from 4.6 to 4.7 g/kg in experiment 1 and 2, respectively. Diets were mixed and pelleted without the use of steam through a 3-mm screen (pellet temperature ≤ 65°C) in the certified feed mill facilities of Hohenheim University's Agricultural Experiment Station, location Lindenhöfe, Eningen, Germany. Representative samples of the diets were taken, pulverized using a laboratory disc mill (Siebtechnik GmbH, Mühlheim an der Ruhr, Germany), and stored at 4°C until chemical analysis. Table 1. Ingredient composition of the diets used in broiler experiments 1 and 2 (g/kg of feed) and calculated nutrient concentration (g/kg DM).1   Experiment 1 (triticale)  Experiment 2 (wheat)    BD  GD20  GD40  BD  GD20  GD40  Ingredient   Triticale genotype  –  200  400  –  –  –   Triticale2  100  100  100  –  –  –   Wheat genotype  –  –  –  –  200  400   Wheat3  –  –  –  100  100  100   Cornstarch  619.7  417.5  215.3  614.8  415.4  214.8   Potato protein  225  225  225  225  225  225   Soybean oil  30  30  30  30  30  30   D,L-Methionine  –  –  –  2.6  0.7  –   L-Arginine  –  –  –  2.4  1.2  –   Limestone  7.3  9.5  11.7  6.7  9.2  11.7   MCPh4  4.5  4.5  4.5  4.5  4.5  4.5   Vitamin premix5  1.5  1.5  1.5  2  2  2   Mineral premix6  1  1  1  1  1  1   Sodium chloride  1  1  1  1  1  1   Choline chloride  2  2  2  2  2  2   Sodium bicarbonate  3  3  3  3  3  3   Titanium dioxide  5  5  5  5  5  5  Calculated nutrient concentration   CP  217  244  272  221  244  268   Crude fat  9  13  16  9  13  17   MJ ME (MJ/kg DM  15.5  15.3  15.2  15.3  15.3  15.3    Experiment 1 (triticale)  Experiment 2 (wheat)    BD  GD20  GD40  BD  GD20  GD40  Ingredient   Triticale genotype  –  200  400  –  –  –   Triticale2  100  100  100  –  –  –   Wheat genotype  –  –  –  –  200  400   Wheat3  –  –  –  100  100  100   Cornstarch  619.7  417.5  215.3  614.8  415.4  214.8   Potato protein  225  225  225  225  225  225   Soybean oil  30  30  30  30  30  30   D,L-Methionine  –  –  –  2.6  0.7  –   L-Arginine  –  –  –  2.4  1.2  –   Limestone  7.3  9.5  11.7  6.7  9.2  11.7   MCPh4  4.5  4.5  4.5  4.5  4.5  4.5   Vitamin premix5  1.5  1.5  1.5  2  2  2   Mineral premix6  1  1  1  1  1  1   Sodium chloride  1  1  1  1  1  1   Choline chloride  2  2  2  2  2  2   Sodium bicarbonate  3  3  3  3  3  3   Titanium dioxide  5  5  5  5  5  5  Calculated nutrient concentration   CP  217  244  272  221  244  268   Crude fat  9  13  16  9  13  17   MJ ME (MJ/kg DM  15.5  15.3  15.2  15.3  15.3  15.3  1BD = basal diet; GD20 = genotype diet supplemented with 20% of 1 of 8 triticale or wheat genotypes; GD40 = genotype diet supplemented with 40% of 1 of 8 triticale or wheat genotypes. 2Mixture of the 8 triticale genotypes used in experiment 1. 3Mixture of the 8 wheat genotypes used in experiment 2. 4MCPh = monocalcium phosphate monohydrate. 5Vitamin premix (Raiffeisenkraftfutterwerke Süd GmbH, Würzburg, Germany) ingredients per kilogram of premix: vitamin A, 6,000,000 IU; vitamin D3, 1,500,000 IU; vitamin E, 15,000 mg; vitamin B1, 1,500 mg; vitamin B2, 3,000 mg; vitamin B6, 3,000 mg; vitamin B12, 15,000 μg; vitamin K3, 1200 mg; nicotinic acid, 25,000 mg; pantothenic acid, 7,000 mg; biotin, 50,000 μg; folic acid, 500 mg. 6Mineral premix (Gelamin SG 1, GFT mbH, Memmingen, Germany) provided per kilogram of complete diet: Mn, 120 mg; Fe, 90 mg; Zn, 80 mg; Cu, 15 mg; I, 1.6 mg; Co, 0.6 mg; Se, 0.5 mg. View Large Table 2. Analyzed phytase activity (FTU/kg DM) and total Ca and P (g/kg DM) of the diets used in broiler experiments 1 (triticale) and 2 (wheat) (g/kg DM of feed).1   BD  GD1  GD2  GD3  GD4  GD5  GD6  GD7  GD8  C (%)  102  20  40  20  40  20  40  20  40  20  40  20  40  20  40  20  40  Experiment 1   Phytase activity3  <100  210  640  480  890  360  850  360  610  360  660  210  550  270  550  230  690   Ca  3.91  4.86  5.71  4.92  5.67  4.89  5.70  4.90  5.71  4.84  5.66  5.04  5.96  4.73  5.96  5.10  5.82   P  2.64  3.35  4.12  3.38  4.28  3.58  4.21  3.45  4.15  3.59  4.22  3.48  4.33  3.37  4.13  3.25  4.05  Experiment 2   Phytase activity3  300  530  1270  660  1080  830  1710  770  1470  680  1160  810  1720  950  1590  510  1120   Ca  3.64  4.46  5.35  4.44  5.45  4.42  5.04  4.41  5.48  4.46  5.54  4.48  5.29  4.55  5.39  4.55  5.45   P  2.50  3.17  4.13  3.28  4.35  3.13  4.20  3.25  4.41  3.31  4.35  3.31  4.14  3.41  4.36  3.22  4.12    BD  GD1  GD2  GD3  GD4  GD5  GD6  GD7  GD8  C (%)  102  20  40  20  40  20  40  20  40  20  40  20  40  20  40  20  40  Experiment 1   Phytase activity3  <100  210  640  480  890  360  850  360  610  360  660  210  550  270  550  230  690   Ca  3.91  4.86  5.71  4.92  5.67  4.89  5.70  4.90  5.71  4.84  5.66  5.04  5.96  4.73  5.96  5.10  5.82   P  2.64  3.35  4.12  3.38  4.28  3.58  4.21  3.45  4.15  3.59  4.22  3.48  4.33  3.37  4.13  3.25  4.05  Experiment 2   Phytase activity3  300  530  1270  660  1080  830  1710  770  1470  680  1160  810  1720  950  1590  510  1120   Ca  3.64  4.46  5.35  4.44  5.45  4.42  5.04  4.41  5.48  4.46  5.54  4.48  5.29  4.55  5.39  4.55  5.45   P  2.50  3.17  4.13  3.28  4.35  3.13  4.20  3.25  4.41  3.31  4.35  3.31  4.14  3.41  4.36  3.22  4.12  1BD = basal diet; GD1–8 = genotype diets supplemented with 20% or 40% of 1 of 8 triticale or wheat genotypes. C = concentration of genotype. 2Mixture of the 8 genotypes of triticale or wheat used in experiment 1 and 2, respectively. 3Determined at pH 5 and 45°C, as described by Greiner and Egli (2003), U/kg. View Large Birds, Animal Management, and Sampling Procedure Two animal experiments were conducted to determine the pcdP of both grains. Experiment 1 investigated triticale and experiment 2 investigated wheat. Both experiments were carried out in the Agricultural Experiment Station of Hohenheim University, location Lindenhöfe, Eningen, Germany, in accordance with German Animal Welfare legislation. All procedures regarding animal handling and treatments were approved by the Animal Welfare Commissioner of the University. Broiler hatchlings (Ross 308, unsexed) were obtained from a local hatchery (Brüterei Süd GmbH and Company KG, Regenstauf, Germany). Birds were raised in floor pens (115 × 115 cm) on wood shavings and fed a commercial starter feed until they were 20 d old (0.90% Ca, 0.65% P, 21.5% CP, 6.3% ether extract, 12.6 MJ ME/kg, and 600 FTU/kg 3-phytase [EC 3.1.3.8, 4a E1600]). In both experiments, birds underwent routine vaccination against coccidiosis (via starter diets) and Newcastle disease on d 12, respectively. On d 20, birds were weighed and randomly allocated to 102 (experiment 1) or 106 floor pens (experiment 2), with 12 birds/pen. In experiment 1 each dietary treatment (BD and 16 triticale GD) was assigned to 6 pens (n = 6 replicates) according to a non-randomized complete block design. In experiment 2, 10 pens received BD treatment (n = 10 replicates), whereas for wheat GD 6 pens (n = 6 replicates) were used. Treatments were arranged in a modified α-design. The α-design is based on a design with 18 treatments tested in 6 complete replicates each with 2 blocks of size 9 within a replicate. Two of these treatments were associated with BD, the other 16 treatments were associated with wheat GDs. In the end, 2 out of 6 observations of the second BD treatment were randomly dropped. In both experiments, pens were arranged in an animal house with 4 columns of 22 to 28 pens per column and 6 sub-columns of 11 to 14 pens per column. Feed and tap water were offered for ad libitum consumption. Experimental diets were fed to animals for 5 or 6 d from d 20 onwards. The average daily feed intake (ADFI) and average daily gain (ADG) were recorded. During the first 2 d, the room temperature was set at 34°C, followed by a subsequent stepwise reduction of 0.5°C per d. Artificial lightening was provided at an intensity of 10 lx. During the first 2 d, light was provided for 24 h; thereafter, provision of light was reduced to 18 h per d. In experiment 1, birds from 4 out of 6 pens per treatment were sacrificed on d 25 of age, whereas samples from the remaining birds were taken on d 26. In experiment 2, all birds were sacrificed at 26 d of age. Animals were stunned with a mixture of 35% CO2, 35% N2, and 30% O2, and euthanized via CO2 asphyxiation. The abdominal cavity of each animal was immediately opened, the digestive tract removed, and the ileum (section between Meckel's diverticulum and 2 cm anterior to the ileo-ceco-colonic junction) dissected. The digesta of the distal half of the ileum was gently flushed out with double-distilled water (4°C) and pooled for all birds per pen into 1 sample per replicate. Samples were immediately frozen at –18°C, freeze-dried (Type Delta 1–24, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode, Germany), ground to pass through a 0.12-mm sieve screen, at a speed of 6,000 rpm (ZM 200 Ultra Centrifugal Mill, Retsch GmbH, Haan, Germany), and stored at 4°C until chemical analysis. Chemical Analyses The DM content of feed and digesta samples was analyzed according to the official methods used in Germany (Verband Deutscher Landwirtschaftlicher Untersuchungs- und Forschungsanstalten [VDLUFA] 1976); Method 3.1). Calcium, P, and Ti in feed and digesta samples were analyzed using an inductively coupled plasma optical emission spectrometer following a sulfuric and nitric acid wet digestion as described by Zeller et al. (2015a). Intrinsic phytase activity of experimental diets was analyzed by the direct incubation method (quantification of liberated inorganic P) at pH 5 and 45°C according to the method described by Greiner and Egli (2003). Calculations and Statistics The body weight (BW), ADG, ADFI, and feed-to-gain ratio were measured on a per-pen basis and adjusted for mortality, which was recorded daily. Prececal digestibility of P (y) from experimental diets was calculated on a pen basis according to the following equation:   \begin{eqnarray} y\left( \% \right) &=& 100 - 100{\rm{ }} \times \ \left( {\frac{{{\rm{Ti}}\,{\rm{in}}\,{\rm{the}}\,{\rm{diet(g/kg\ }}\,{\rm{DM)}}}}{{{\rm{Ti}}\,{\rm{in}}\,{\rm{the}}\,{\rm{digesta(g/kg}}\,{\rm{DM)}}}}} \right)\nonumber\\ && \times \left( {\frac{{{\rm{P}}\,{\rm{in}}\,{\rm{the}}\,{\rm{digesta(g/kg}}\,{\rm{DM)}}}}{{{\rm{P}}\,{\rm{in}}\,{\rm{the}}\,{\rm{diet(g/kg}}\,{\rm{DM)}}}}} \right) \end{eqnarray} (1)The amount of pcdP (mg/d) was calculated by multiplying the daily total P intake with diet (mg/d) and the respective digestibility (%), divided by 100. In 1 of the 6 pens receiving BD treatment, implausible performance and pcdP data (negative values, which differed twice the SD from means) were generated, and the results from this pen were not included in the data analysis. Statistical analyses were performed using the software package SAS for Windows (Version 9.3, SAS Institute, Cary, NC). Performance data and pcdP values of experimental diets were analyzed using a mixed model approach (procedure PROC MIXED) considering the treatment factors diet type (BD vs. GD), diet, and concentration of genotype as fixed factors and effects of block (just in experiment 2) and replicate as random effects. If the model fit was improved, additional random effects of “column” and “sub-column” was considered in the model. These factors were included via post-blocking according to the allocation of pens in the animal house. The model can be described by:   \begin{eqnarray} {y_{ijklmno}} &=& \mu + {\alpha _i} + {\beta _{ij}} + {\gamma _{ik}} + {\left( {\beta \gamma } \right)_{ijk}} + re{p_l} + {b_{lm}}\nonumber\\ && +\, {c_n} + {s_{no}}_{} + {e_{ijklmno}}, \end{eqnarray} (2)where μ = general mean; αi= effect of the ith diet type; βj = effect of the jth diet within diet type; γk = effect of the kth concentration of genotype within diet type; (βγ)jk = interaction effect of the jth diet and kth concentration of genotype within diet type; repl = effect of the lth replicate, blm = effect of the random mth block; cn = random effect of the nth column; sno = random effect of the oth sub-column within the nth column, and eijklmno = error of observation yijklmno. Block effects were only fitted for experiment 2. To ensure normal distribution and variance homogeneity of residuals, data on pcdP (%) were subjected to arcsine square-root transformation prior to analysis. Least-square means from the analysis were back-transformed for presentation only. A multiple t-test for treatment comparisons was applied only after a significant F-test. The level of significance was set at α = 0.05. Prececal digestibility of P from triticale genotypes was calculated using a mixed-model approach similar to that used to determine the prececal digestibility of amino acids from cereal grains, recently published by Zuber et al. (2016). This approach implies there is a linear relationship between P intake and digested P within the range of P intakes. The model used for triticale genotypes can be described as follows:   \begin{equation}{y_{ilmno}} = \mu + r \times {\beta _i} + re{p_l} + {b_{lm}} + {c_n} + {s_{no}} + {e_{ilmno}},\end{equation} (3)where: μ = intercept (representing the digestibility of the BD); r = ratio of the daily P intake attributable to the triticale genotype and the daily P intake attributable to the BD; βi = regression coefficient of genotype i; repl = effect of the lth replicate; blm = random effect of the mth block; cn = random effect of the nth column; sno = random effect of the oth sub-column, and eilmno = error of yilmno. The slope βi between r and yilmno(βi) represents the P digestibility of triticale genotype i. Regression coefficients were calculated simultaneously using the PROC MIXED procedure of SAS and compared between genotypes via contrasts using the ESTIMATE statement of the PROC MIXED procedure. Although diets were low in P, the assumption of a linear relationship between P intake and pcdP (mg/d) could not be confirmed in experiment 2. Thus, model (3) was adapted to fit separate slopes for both concentrations of all 8 wheat genotypes (in total 16 slopes) to estimate the pcdP (%). Relationships between pcdP and different chemical or physical characteristics of triticale and wheat genotypes as previously reported (Rodehutscord et al., 2016) were examined by regressing them to P digestibility values βi. The following regression was assumed:   \begin{equation} {\beta _i} = \pi + \tau \times {x_i} + {\varepsilon_i},\end{equation} (4)where π corresponds to a general digestibility, τ is the slope parameter between digestibility and chemical or physical property measure xi for ith genotype and εi is the random genotype specific deviation of digestibility of genotype i from the regression line. A significant test of τ corresponds to a relationship between digestibility and the corresponding property. To estimate τ, βi in (3) was replaced with (4) and the resulting model was re-run. Note that βi was multiplied with r in (3), thus each of the 3 effects in (4) is multiplied with r. As 92 properties were tested for relationship in both grains, we account for multiple testing by a Bonferroni correction of α. RESULTS Experiment 1 (triticale) As expected, the diet type (BD vs. GD) had a significant (P < 0.05) effect on the performance of birds and on the pcdP of the experimental diets (Table 3). Moreover, the concentration of triticale genotypes had a significant (P < 0.05) effect on performance data. The increased ADG (and decreased feed: gain ratio indicated that birds fed the GD performed better than those fed the BD. Additionally, the birds’ performance was improved by increasing the concentration of triticale genotypes in the diet from 20% to 40%. Table 3. BW1, ADG1, ADFI1, P intake1, feed-to-gain ratio1 and pcdP of broilers fed basal diet (BD) or diets containing different concentrations (C) of triticale genotypes (GD1–8) in experiment 1.2 Effect  BW (g)  ADG (g/d)  ADFI (g/d)  Feed:gain (g/g)  P intake (mg/d)  pcdP (mg/d)  pcdP (%)  Diet type3   BD  942  8  59  9.00  142  53  36.5   GD  1030  23  79  3.77  276  146  52.7   SEM  18.3  1.7  2.5  0.551  8.6  8.7  2.90   P value  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  Diet (Diet type)   GD1  1024  19  77  4.85  264  133  51.1   GD2  1025  22  82  3.91  287  153  53.5   GD3  1022  24  78  3.50  278  150  53.4   GD4  1027  23  76  3.64  265  126  46.7   GD5  1048  25  82  3.42  294  161  55.0   GD6  1010  23  77  3.64  276  151  54.7   GD7  1043  25  79  3.50  270  146  54.2   GD8  1042  23  81  3.67  271  145  52.8   SEM  17.1  1.57  2.3  0.503  8.0  7.9  2.67   P value  0.423  0.233  0.099  0.540  0.008  0.022  0.389  C (Diet type)   GD20  1012  19  75  4.33  236  122  51.7   GD40  1049  27  83  3.20  315  170  53.6   SEM  13.1  0.8  1.7  0.252  6.0  4.9  1.40   P value  <0.001  <0.001  <0.001  0.002  <0.001  <0.001  0.312  Diet × C (Diet type)   P value  0.228  0.475  0.617  0.810  0.562  0.509  0.506  Effect  BW (g)  ADG (g/d)  ADFI (g/d)  Feed:gain (g/g)  P intake (mg/d)  pcdP (mg/d)  pcdP (%)  Diet type3   BD  942  8  59  9.00  142  53  36.5   GD  1030  23  79  3.77  276  146  52.7   SEM  18.3  1.7  2.5  0.551  8.6  8.7  2.90   P value  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  Diet (Diet type)   GD1  1024  19  77  4.85  264  133  51.1   GD2  1025  22  82  3.91  287  153  53.5   GD3  1022  24  78  3.50  278  150  53.4   GD4  1027  23  76  3.64  265  126  46.7   GD5  1048  25  82  3.42  294  161  55.0   GD6  1010  23  77  3.64  276  151  54.7   GD7  1043  25  79  3.50  270  146  54.2   GD8  1042  23  81  3.67  271  145  52.8   SEM  17.1  1.57  2.3  0.503  8.0  7.9  2.67   P value  0.423  0.233  0.099  0.540  0.008  0.022  0.389  C (Diet type)   GD20  1012  19  75  4.33  236  122  51.7   GD40  1049  27  83  3.20  315  170  53.6   SEM  13.1  0.8  1.7  0.252  6.0  4.9  1.40   P value  <0.001  <0.001  <0.001  0.002  <0.001  <0.001  0.312  Diet × C (Diet type)   P value  0.228  0.475  0.617  0.810  0.562  0.509  0.506  1Average of birds slaughtered 25 and 26 d post hatch. 2Data are given as LS means. BD had n = 5 and GD1–8 had n = 6 pens per treatment with 10 birds per pen (GD320 and GD640: BW gain and F: G had n = 5). 3Diet type = BD vs. GD. View Large The pcdP of the BD was on average (standard error [SE] in parentheses) 36% (4.0%), and was 17% lower than that of the GD (54% [3.7%]). Neither the concentration of genotypes nor the diet itself affected the pcdP (%) of GD. However, P intake and the pcdP (mg/d) significantly (P < 0.05) differed between diets, with highest values found for GD2 and GD5 and the lowest for GD1 and GD4. No interaction was found between diet and concentration of triticale genotypes on the performance of birds or on the pcdP of experimental diets. The pcdP of triticale genotypes estimated by linear regression according to model 3 varied between triticale genotypes and ranged from 53% to 78% (Table 4). The pcdP for genotype no. 4 (53%) was significantly (P < 0.05) lower compared with genotype no. 3, 5, 6, and 8 (75%, 74%, 73%, 78%). A significant correlation was not observed between the chemical and physical properties of genotypes and their pcdP. Table 4. Estimated digestibility (%) and SE of the triticale genotypes fed to broilers in experiment 1.1   Triticale genotype2      1  2  3  4  5  6  7  8  P value3  Estimate  63.7a,b  72.0a,b  74.6a  52.9b  74.2a  73.2a  71.1a,b  78.2a                      <0.001  SE  13.40  9.40  9.39  9.97  9.32  9.02  10.25  10.98      Triticale genotype2      1  2  3  4  5  6  7  8  P value3  Estimate  63.7a,b  72.0a,b  74.6a  52.9b  74.2a  73.2a  71.1a,b  78.2a                      <0.001  SE  13.40  9.40  9.39  9.97  9.32  9.02  10.25  10.98    1Genotypes 1–8 had n = 6 pens per treatment with 10 birds per pen. 2The slopes (βi) between r and yijkl from equation 3 multiplied by 100 represent the digestibility of the respective triticale genotype i. 3Estimates within a row not sharing a common superscript differ significantly (multiple t-tests in case of interaction), P ≤ 0.05. View Large Experiment 2 (wheat) Similar to experiment 1, diet type had a significant (P < 0.05) effect on the birds’ performance and on the pcdP of diets in experiment 2. In addition, similar to experiment 1, feeding of GD increased the BW of birds at d 26 of age, ADFI and ADG, and decreased the feed: gain ratio compared with BD (Table 5). The P intake and amount of pcdP (mg/d) also increased in animals fed the GD instead of the BD; however, in contrast to experiment 1, the average (SE) pcdP (%) decreased from 63% (1.5%) in the BD to 55% (1.83%) in the GD. Table 5. BW at d 26 of age, ADG, ADFI, P intake, feed-to-gain ratio, and pcdP of broilers fed basal diet (BD) or diets containing different concentrations (C) of wheat genotypes (GD1–8) in experiment 2.1 Diet  BW (g)  ADG (g/d)  ADFI (g/d)  Feed:gain (g/g)  P intake (mg/d)  pcdP (mg/d)2  pcdP (%)  BD  987  19  83  5.17  188  118  63.1  GD120  1039  28  100  3.64  287  180  62.8  GD140  1027  25  101  4.26  374  196  52.9  GD220  1025  27  96  3.62  285  173b  60.7  GD240  1004  24  96  4.10  374  195a  52.2  GD320  1026  25  93  3.78  262  154b  58.4  GD340  1014  21  104  4.97  391  201a  51.7  GD420  1018  28  103  3.72  301  173b  57.7  GD440  1037  30  106  3.70  417  210a  50.1  GD520  1046  31  100  3.31  297  192  64.7  GD540  1026  24  99  4.29  385  185  48.0  GD620  1052  29  108  3.71  323  191  59.0  GD640  1018  24  100  4.24  375  171  46.0  GD720  1028  27  102  3.81  313  193b  62.0  GD740  1055  30  105  3.59  413  217a  52.4  GD820  1024  27  98  3.91  286  168  58.4  GD840  1025  24  100  4.67  370  177  48.0  Pooled SEM2  17.2 (13.7)  2.1 (1.7)  3.8 (3.0)  0.413 (0.320)  11.9 (9.5)  8.4 (6.9)  1.83 (1.46)  ANOVA P value3  Diet type4  0.003  <0.001  <0.001  0.005  <0.001  <0.001  <0.001  Diet (Diet type)  0.778  0.204  0.212  0.633  0.004  0.003  0.007  C (Diet type)  0.440  0.022  0.431  0.011  <0.001  <0.001  <0.001  Diet × C (Diet type)  0.602  0.327  0.375  0.711  0.054  0.001  0.088  Diet  BW (g)  ADG (g/d)  ADFI (g/d)  Feed:gain (g/g)  P intake (mg/d)  pcdP (mg/d)2  pcdP (%)  BD  987  19  83  5.17  188  118  63.1  GD120  1039  28  100  3.64  287  180  62.8  GD140  1027  25  101  4.26  374  196  52.9  GD220  1025  27  96  3.62  285  173b  60.7  GD240  1004  24  96  4.10  374  195a  52.2  GD320  1026  25  93  3.78  262  154b  58.4  GD340  1014  21  104  4.97  391  201a  51.7  GD420  1018  28  103  3.72  301  173b  57.7  GD440  1037  30  106  3.70  417  210a  50.1  GD520  1046  31  100  3.31  297  192  64.7  GD540  1026  24  99  4.29  385  185  48.0  GD620  1052  29  108  3.71  323  191  59.0  GD640  1018  24  100  4.24  375  171  46.0  GD720  1028  27  102  3.81  313  193b  62.0  GD740  1055  30  105  3.59  413  217a  52.4  GD820  1024  27  98  3.91  286  168  58.4  GD840  1025  24  100  4.67  370  177  48.0  Pooled SEM2  17.2 (13.7)  2.1 (1.7)  3.8 (3.0)  0.413 (0.320)  11.9 (9.5)  8.4 (6.9)  1.83 (1.46)  ANOVA P value3  Diet type4  0.003  <0.001  <0.001  0.005  <0.001  <0.001  <0.001  Diet (Diet type)  0.778  0.204  0.212  0.633  0.004  0.003  0.007  C (Diet type)  0.440  0.022  0.431  0.011  <0.001  <0.001  <0.001  Diet × C (Diet type)  0.602  0.327  0.375  0.711  0.054  0.001  0.088  1Data are given as LS means. BD had n = 10 and GD1–8 had n = 6 pens per treatment with 12 birds per pen. 2Pooled SEM of GD (SEM of BD). 3Estimates within a GD having different superscripts differ significantly among the 2 concentrations of genotypes tested (multiple t-tests in case of interaction), P ≤ 0.05; due to the large number of possible comparisons we refer only to those differences between concentrations of wheat within a genotype. 4Diet type = BD vs. GD. View Large Increasing the concentration of wheat from 20% to 40% had a negative effect (P < 0.05) on the ADG and feed: gain ratio. Animals fed the GD40 showed a lower ADG (25 vs. 28 g/d) and a higher feed: gain ratio (4.2 vs. 3.7 g/g) than those fed the GD20. A higher concentration of wheat genotypes had a negative effect (P < 0.05) on the pcdP (%) of diets, which decreased from 60% (1.0%) in GD20 to 50% (1.0%) in GD40. Moreover, a significant effect of diet was observed on the pcdP (%). While GD1 and GD7 showed the highest pcdP (58% and 57%), significantly (P < 0.05) lower values were found for GD6 and GD8 (53%). In addition, the former diets also showed the highest ADG and lowest feed: gain ratio, but the lowest ADG and highest feed: gain ratio was observed for GD3. An interaction between diet and wheat genotype concentration was detected only for pcdP (mg/d). Thus, the results showed a significant (P < 0.05) increase in the amount of pcdP with the higher wheat genotype concentration (40%) in GD2, GD3, GD4, and GD7, whereas no differences between concentrations were found for the other GD. Estimates of the pcdP of wheat genotypes obtained by regression analysis are given in Table 6. The values ranged from 38% to 67% with inclusion of 20% wheat genotypes and from 20% to 38% with inclusion of 40% wheat genotypes. At the concentration of 20%, genotypes no. 1 and 5 showed the highest pcdP, which was significantly (P < 0.05) different from that of genotypes no. 3, 4, 6, and 8. At the concentration of 40%, the pcdP was highest for genotypes no. 1, 2, and 7, differing significantly (P < 0.05) from genotype no. 6, which showed the lowest pcdP. A significant (P < 0.05) decrease in the pcdP with increased wheat concentration was observed for all genotypes except for no. 3, 4, and 8. As for triticale genotypes, significant correlations were not observed between the chemical and physical properties of genotypes and their pcdP. Table 6. Estimated digestibility (%) and SE at different inclusion concentrations (C, %) of wheat genotypes contained in diets fed to broilers in experiment 2.1     Wheat genotype2      C  1  2  3  4  5  6  7  8  P value3  Estimate  20  59.4A,a,b  51.5A,bd  38.1d  39.5d  66.9A,a  43.4A,c,d  56.3A,ac  38.7d    SE    5.60  4.97  5.40  4.80  5.00  5.60  4.90  5.70                        <0.001  Estimate  40  36.7B,a  36.7B,a  34.7a,b  32.1a,b  28.5B,a,b  20.2B,b  38.0B,a  23.8a,b    SE    7.10  6.00  7.20  6.22  5.70  6.00  5.60  6.30        Wheat genotype2      C  1  2  3  4  5  6  7  8  P value3  Estimate  20  59.4A,a,b  51.5A,bd  38.1d  39.5d  66.9A,a  43.4A,c,d  56.3A,ac  38.7d    SE    5.60  4.97  5.40  4.80  5.00  5.60  4.90  5.70                        <0.001  Estimate  40  36.7B,a  36.7B,a  34.7a,b  32.1a,b  28.5B,a,b  20.2B,b  38.0B,a  23.8a,b    SE    7.10  6.00  7.20  6.22  5.70  6.00  5.60  6.30    1Genotypes 1–8 had n = 6 pens per treatment with 12 birds per pen. 2The slopes (βi) between r and yijkl from equation 3 multiplied by 100 represent the digestibility of the respective wheat genotype i at concentration of 20% or 40%. 3Estimates within a row not sharing a common lower case or within a column not sharing a common capital letter differ significantly (multiple t-tests in case of interaction), P ≤ 0.05. View Large DISCUSSION A standard trial protocol for determination of digestible P from different P sources is an important prerequisite for the harmonization of P evaluation and requirement standards for poultry (WPSA, 2013). In a future table of digestible P in feedstuffs data from the present study can contribute information on triticale and wheat. Previous studies based on the WPSA protocol have considered rapeseed meal (Olukosi et al., 2015), wheat distillers’ grains with solubles (Adebiyi and Olukosi, 2015), soybean meal (Rodehutscord et al., 2017), corn and canola meal (Mutucumarana et al., 2014b), and corn and wheat (Kupcikova et al., 2017). Effect of Triticale Genotype and its Concentration on Prececal P Digestibility The results of our experiments confirmed the hypothesis that the triticale or wheat genotype affects the pcdP of grains in broiler chickens. However, compared with wheat, the pcdP of triticale genotypes was less variable, ranging between 53% and 78%, with most of the triticale genotypes showing a value higher than 70%. Thus, most genotypes did not differ in their pcdP. This explains the missing effect of diet on the pcdP in GD, in which a maximum of one-third of total P comes from the test grain. These results are consistent with those of previous studies on broiler chickens fed diets containing 45% of 4 different triticale varieties, in which the variety had no effect on the ash content of tibiotarsi (Jondreville et al., 2007). The average (SE) pcdP of GD with triticale was 54% (3.7%) in the present study. This value is consistent with the results of Jamroz et al. (1996) and Jamroz et al. (1998), who detected a P retention of 46% and 51% from diets containing 55% or 40% triticale, respectively, in 6-wk-old broilers. No correlations between the pcdP and chemical or physical properties of triticale genotypes were detected in the present study. Jondreville et al. (2007) described a positive response of bone ash contents to intrinsic phytase of triticale in broiler chickens. However, consistent with our findings, those authors were unable to detect differences between diets with phytase activity of 620 to 1,390 U/kg mainly derived from triticale varieties with activities of 768 to 1,700 U/kg of grain. Moreover, our results revealed no effect of diet extract viscosity on the pcdP of triticale genotypes, although mineral absorption in broiler chickens from wheat- (van der Klis et al., 1995) or corn-based diets (Mohanna et al., 1999) has been shown to be impaired by increased diet viscosity. The extract viscosity of triticale genotypes investigated in the present study ranged only between 1.10 and 1.44 mPa·s (at shear rate of 380/s; Rodehutscord et al., 2016), which probably represents the variation being too low to allow any relationship between viscosity and pcdP of genotypes to be detected. In broiler chickens fed P-deficient diets based on different triticale varieties with viscosities of 1.79 to 2.7 mL/g, there was no difference in bone ash contents and variety did not affect animal performance (Jondreville et al., 2007). The latter was also observed in the present study and by Elangovan et al. (2011) with non P-deficient diets. While the increased amount of pcdP (mg/d) with higher P contents in triticale genotypes did not affect animal performance, the increased amount of pcdP (mg/d) with GD40 compared to GD20 was coincided with a higher BW, ADG, ADFI and feed to gain ratio. However, the positive effect of higher triticale concentrations on animal performance may be not only a result of increased pcdP (mg/d) as overall nutrient composition of GD40 (Table 1) changed with substitution of corn starch by further triticale. Effect of Wheat Genotype and its Concentration on Prececal P Digestibility For wheat-based diets, the pcdP of the GD ranged between 53% and 58%. These results are consistent with those of previous studies in broiler in which for diets based on different wheat varieties, P retention of 45% to 64% (Barrier-Guillot et al., 1996) and a pcdP of 47% to 55% (van der Klis et al., 1995) were reported. However, the differences found in the pcdP (%) or the amount of pcdP (mg/d) in the GD1–8 diets, did not affect animal performance in the present study. Similar to triticale, the concentration of wheat was more important with respect to animal performance than the diet itself. However, in contrast to experiment 1, the increase in wheat from 20% to 40% considerably decreased the ADG and increased the feed: gain ratio, although the amount of pcdP (mg/d) was either similar or increased with 40% wheat. Thus, factors other than the digestible P must have been of higher relevance for animal performance under the given experimental conditions. In the present study, a 10% decrease in the pcdP was observed with increased wheat inclusion; however, Mutucumarana et al. (2014a) found no effect of wheat concentration on the pcdP in 4-wk-old broiler chickens fed diets containing 24% to 95% wheat. Moreover, these authors reported a positive effect of increasing wheat proportions on animal performance. However, the semi-synthetic diets used by Mutucumarana et al. (2014a) contained very low P contents at 0.85 to 3.08 g total P/kg, and showed a pcdP of only 42%. Thus, animal performance was even lower than that reported in the present study, which may explain the different response of animals to wheat concentration. The pcdP of the test wheat in the experiment of Mutucumarana et al. (2014a) was calculated to be 46%. From diets based on 99% (Wu et al., 2004) or 94% (Rutherfurd et al., 2002) wheat, a pcdP of 51% or 50% was reported for 5-wk-old broiler chickens, respectively. These values are in the range of the pcdP determined for wheat genotypes at the concentration of 20% in the present study. However, our results indicated that pcdP was reduced by 3% to 38% when the wheat concentration was increased from 20% to 40%. In contrast, Mutucumarana et al. (2014a) described a linear relationship between dietary P content and P output in digesta samples in the range of 24% to 95% of dietary wheat. In their study, the within-treatment variation considerably increased with wheat concentrations higher than 24%. As a result, linear regression analysis for the wheat tested by Mutucumarana et al. (2014a) showed a reduced goodness-of-fit than has been reported for other P sources, such as corn, canola meal (Mutucumarana et al., 2014a), and soybean meal (Rodehutscord et al., 2017). Thus, it remains unclear whether the amount of pcdP from wheat linearly increases with increasing dietary P derived from wheat in growing broiler chickens. Our data, which were obtained from several wheat genotypes and were based on more observations than used by Mutucumarana et al. (2014a), do not confirm linearity. A recent digestibility ring test revealed great differences in the results between institutions that determined the pcdP of soybean meal (Rodehutscord et al., 2017). The authors hypothesized factors like feeding and housing in the pre-experimental period might influence the degradation of phytate P and pcdP. Thus, comparisons between P digestibility studies, even if conducted under similar experimental conditions, should be made with great caution until the WPSA protocol is more refined. In the present study, correlation analysis revealed no relationship between total or phytate P contents in wheat genotypes and the pcdP of test wheats. These results are in accordance with those of former studies (Barrier-Guillot et al., 1996), in which total and phytate P contents of different wheat varieties were not related to the P retention of broilers from wheat-based diets. Additionally, no correlation was detected between the intrinsic phytase activity of wheat genotypes and the pcdP. In contrast, Barrier-Guillot et al. (1996) reported a positive correlation between the phytase activity of wheat varieties and P retention of broilers. However, when considering from their data only the wheat varieties that had been grown under similar agronomic conditions, no correlation could be found between phytase activity and P retention. Moreover, a minor effect of the intrinsic phytase activity in wheat on broiler chickens’ pcdP was suggested by Shastak et al. (2014), Zeller et al. (2015b), and Leytem et al. (2008a, b). Additionally, no effect of the extract viscosity of wheat genotypes was observed on the pcdP in the present study. In contrast, van der Klis et al. (1995) reported a decreased pcdP from corn-wheat-based diets with increasing extract viscosity of the different wheat varieties fed to broiler chickens. However, although supplementation of xylanase decreased the viscosity of the jejunal and ileal digesta in the studies of van der Klis et al. (1995), the enzyme did not increase the pcdP from diets. Thus, factors other than digesta viscosity must have been more important for the P digestibility. Zuber and Rodehutscord (2016) determined the digestibility of amino acids in laying hens from 20 different wheat genotypes, including the 8 genotypes used in the present study. As found in our study, neither physical nor chemical properties sufficiently explained the differences observed between genotypes. These authors hypothesized that differences in the fine structure, substitution pattern, or structural arrangement of NSP may affect solubility and other properties of polymers in the gastrointestinal tract, which in turn, can influence the digestibility of nutrients, but cannot be described by the method used for carbohydrate analysis of genotypes in the present study. To conclude, the results of the present study confirm our hypothesis that P digestibility from triticale and wheat in broiler chickens is influenced by genotype. The physical and chemical properties used for characterization of triticale and wheat genotypes did not explain the differences found in P digestibility. However, our results confirm the prior observation that total or phytate P contents as well as the intrinsic phytase activity are of minor importance for the pcdP of grains in broiler chickens. Moreover, our data suggest higher P digestibility from triticale than from wheat genotypes, and a different response of animals to the concentration of both grains. The factors contributing to differences in the P digestibility between grains and genotypes remain unclear. ACKNOWLEDGEMENTS The project was supported by funds from the Federal Ministry of Food, Agriculture, and Consumer Protection (BMELV) based on a decision of the Parliament of the Federal Republic of Germany via the Federal Office for Agriculture and Food (BLE) under the innovation support program. Contributions to the project made by Margit Schollenberger, Helga Ott and Helga Terry are also gratefully acknowledged. REFERENCES Abelson P. H. 1999. A potential phosphate crisis. Science  283. Adebiyi A., Olukosi O.. 2015. 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Variability of prececal phosphorus digestibility of triticale and wheat in broiler chickens

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Abstract

Abstract The aim of this study was to evaluate the P digestibility of different wheat and triticale genotypes in growing broiler chickens. Additionally, the relationships between physical or chemical properties of genotypes and P digestibility were determined. A low P, low Ca basal diet based on cornstarch and soybean meal was supplemented with 20% or 40% of 8 different wheat or triticale genotypes at the expense of cornstarch. Experimental diets were fed to broilers between d 20 and 26 of age before digesta samples were collected from their lower ileum for determination of prececal P digestibility (pcdP). Triticale-based diets had an average pcdP of 54%. Neither the concentration of triticale nor the genotype itself affected the pcdP of diets. The pcdP of triticale genotypes calculated by linear regression analysis ranged from 53% to 78%. No correlations were found between physical or chemical properties (viscosity, phytase activity, total and phytate P contents) and the pcdP of triticale genotypes. In contrast, a significant (P < 0.05) effect of genotype and wheat concentration was observed in wheat-based diets. The average pcdP of diets decreased from 60% to 50% by increasing the wheat concentration from 20% to 40%. As no linear relationship was observed between P intake and the amount of pcdP in the diet, the pcdP (%) of wheat genotypes was calculated separately for both concentrations, and accounted for 38% to 67% and 20% to 38% with 20% and 40% wheat inclusion in diets, respectively. Single chemical and physical characteristics could not explain the observed differences in P digestibility. Our results revealed a high variation in the pcdP of different wheat and triticale genotypes that should be considered in diet formulation. However, further research is needed to identify factors that contribute to variation of P digestibility in both grains. INTRODUCTION Phosphorus (P) is an element with special relevance for sustainable poultry feeding. In non-ruminants, the digestibility of plant P bound to phytate, a salt of phytic acid that represents the main storage form of plant P, is limited due to the lack of sufficient production of endogenous phytase in the upper gastrointestinal tract. Thus, diets are usually supplemented with highly digestible feed phosphates or phytase to meet the animal's P requirement. However, these supplements are expensive and global resources of raw phosphates are limited (Abelson, 1999). Moreover, excessive P intake can be detrimental to the environment in regions of high livestock density (Sharpley, 1999; Rodehutscord, 2008). Thus, for a responsible handling of P sources and a precise supply of dietary P to animals a sound knowledge on different feed raw materials, which differ considerably in their P digestibility, is required (World's Poultry Science Association [WPSA], 2013). Therefore, the WPSA recently published a standard protocol for testing the P digestibility of feed raw materials (WPSA, 2013). Wheat is an important grain in poultry nutrition (Coskuntuna et al., 2008; Stef et al., 2013) and is generally included at up to 55% in broiler diets (Gutierrez et al., 2009). Among cereals, wheat contains moderate total P (∼3.7 g/kg dry matter [DM]) and phytate P (∼1.9 g/kg DM) concentrations, while its intrinsic phytase activity is high (∼1,850 units/kg DM) (Rodehutscord et al., 2016). Thus, wheat can contribute considerably to an animal's P supply. However, in the only studies reported to date that have investigated wheat using the WPSA (2013) protocol, the prececal P digestibility (pcdP) of wheat was 18% and 46% in 4-5 wk-old broilers (Mutucumarana et al., 2014a; Kupcikova et al., 2017). Further reports in the literature refer only to the pcdP or P retention from wheat-based complete diets (van der Klis et al., 1995; Rutherfurd et al., 2002; Wu et al., 2004; Afsharmanesh et al., 2008). Afsharmanesh et al. (2008) used 3-wk-old broilers and fed diets based on 80% Durum or hard red spring wheat and found pcdP values of 60% and 64%, respectively, thus indicating a significant effect of the wheat variety on the pcdP. Different wheat varieties may differ in contents of total P, phytate P, soluble and insoluble non-starch polysaccharides (NSP) and their intrinsic phytase activity and extract viscosity (van der Klis et al., 1995; Zyla et al., 1999; Erdal et al., 2002; Steiner et al., 2007; Rodehutscord et al., 2016) which may affect the pcdP in vivo (van der Klis et al., 1995) and the release of inorganic P in vitro (Zyla et al., 1999). Triticale, an intergenic hybrid, is an alternative cereal grain combining the feeding characteristics of wheat and winter hardiness, drought, and disease resistance of rye (Boros, 1999). Thus, triticale is more important in regions where wheat or corn cannot be grown (Djekic et al., 2011). Modern high-yielding varieties with improved nutritional properties have the potential to replace wheat and corn in poultry diets (Widodo et al., 2015). However, more research is necessary to evaluate the nutritive value of triticale, especially with respect to its P digestibility. On average, concentrations of phytate P, total and soluble NSP are similar to those found in wheat, while the total P content, intrinsic phytase activity, and extract viscosity are higher in triticale than in wheat. However, the chemical composition of different triticale varieties may vary considerably (Jondreville et al., 2007; Rodehutscord et al., 2016). Hence, the increasing use of triticale and the differences found between varieties necessitate an assessment of the different varieties’ P digestibility in poultry. The objective of the present study was to determine the pcdP of different wheat and triticale genotypes grown under similar environmental and agronomic conditions using the protocol recommended by the WPSA (2013). Moreover, the relationships between physical or chemical properties of grains and the pcdP of genotypes were investigated. MATERIALS AND METHODS This study was part of the collaborative research project referred to as GrainUp (www.grain-up.de). The 8 triticale and 8 wheat genotypes used in this study were grown under similar environmental and agronomic conditions, but specific for each species. Cultivation and processing, as well as physical and chemical properties of these genotypes are described in detail elsewhere (Rodehutscord et al., 2016). Genotypes used in this study were numbered in the same way as in Rodehutscord et al. (2016). The methodological approach used in this study followed the protocol recommended to determine the P digestibility in poultry from plant sources (WPSA, 2013). Experimental Diets Two cornstarch-potato protein-based basal diets (BD) were formulated to contain adequate concentrations of all nutrients according to the recommendations of the Gesellschaft für Ernährungsphysiologie (GfE, 1999), with the exceptions of Ca and P (Table 1). The 2 BD contained either 10% of a mixture of the 8 triticale genotypes (experiment 1) or 10% of a mixture of the 8 wheat genotypes (experiment 2) tested in this study. In 32 further experimental diets (genotype diets, GD), each of the 8 triticale and wheat genotypes was added to the BD at 2 different concentrations (20% or 40%, Table 1) at the expense of cornstarch, thus making the triticale or wheat genotype the only source of variation in P content of the diets. Prior to mixing, cereal grains were ground to pass through a 2-mm sieve screen. Limestone was also added to the GD at the expense of cornstarch to retain a constant Ca: P ratio in all diets. Titanium dioxide was included (0.5%) as an indigestible marker. Analyzed phytase activity and concentrations of total P and Ca of the experimental diets are presented in Table 2. Phytase activity ranged from <100 to 890 FTU/kg DM in triticale-based diets and from 300 to 1720 FTU/kg DM in wheat-based diets. The concentrations of total P in experimental diets with triticale ranged from 2.64 to 4.33 and from 2.5 to 4.36 g/kg DM in diets with wheat. The Ca: total P ratio in diets with triticale or wheat was on average (standard deviation [SD] in parentheses) 1.41:1.0 (0.06) and 1.33:1.0 (0.07), respectively, which was very close to the ratio (1.3:1.0 to 1.4:1.0) recommended in the WPSA protocol (WPSA, 2013). Analyzed titanium dioxide concentrations in diets ranged from 4.8 to 4.9 g/kg and from 4.6 to 4.7 g/kg in experiment 1 and 2, respectively. Diets were mixed and pelleted without the use of steam through a 3-mm screen (pellet temperature ≤ 65°C) in the certified feed mill facilities of Hohenheim University's Agricultural Experiment Station, location Lindenhöfe, Eningen, Germany. Representative samples of the diets were taken, pulverized using a laboratory disc mill (Siebtechnik GmbH, Mühlheim an der Ruhr, Germany), and stored at 4°C until chemical analysis. Table 1. Ingredient composition of the diets used in broiler experiments 1 and 2 (g/kg of feed) and calculated nutrient concentration (g/kg DM).1   Experiment 1 (triticale)  Experiment 2 (wheat)    BD  GD20  GD40  BD  GD20  GD40  Ingredient   Triticale genotype  –  200  400  –  –  –   Triticale2  100  100  100  –  –  –   Wheat genotype  –  –  –  –  200  400   Wheat3  –  –  –  100  100  100   Cornstarch  619.7  417.5  215.3  614.8  415.4  214.8   Potato protein  225  225  225  225  225  225   Soybean oil  30  30  30  30  30  30   D,L-Methionine  –  –  –  2.6  0.7  –   L-Arginine  –  –  –  2.4  1.2  –   Limestone  7.3  9.5  11.7  6.7  9.2  11.7   MCPh4  4.5  4.5  4.5  4.5  4.5  4.5   Vitamin premix5  1.5  1.5  1.5  2  2  2   Mineral premix6  1  1  1  1  1  1   Sodium chloride  1  1  1  1  1  1   Choline chloride  2  2  2  2  2  2   Sodium bicarbonate  3  3  3  3  3  3   Titanium dioxide  5  5  5  5  5  5  Calculated nutrient concentration   CP  217  244  272  221  244  268   Crude fat  9  13  16  9  13  17   MJ ME (MJ/kg DM  15.5  15.3  15.2  15.3  15.3  15.3    Experiment 1 (triticale)  Experiment 2 (wheat)    BD  GD20  GD40  BD  GD20  GD40  Ingredient   Triticale genotype  –  200  400  –  –  –   Triticale2  100  100  100  –  –  –   Wheat genotype  –  –  –  –  200  400   Wheat3  –  –  –  100  100  100   Cornstarch  619.7  417.5  215.3  614.8  415.4  214.8   Potato protein  225  225  225  225  225  225   Soybean oil  30  30  30  30  30  30   D,L-Methionine  –  –  –  2.6  0.7  –   L-Arginine  –  –  –  2.4  1.2  –   Limestone  7.3  9.5  11.7  6.7  9.2  11.7   MCPh4  4.5  4.5  4.5  4.5  4.5  4.5   Vitamin premix5  1.5  1.5  1.5  2  2  2   Mineral premix6  1  1  1  1  1  1   Sodium chloride  1  1  1  1  1  1   Choline chloride  2  2  2  2  2  2   Sodium bicarbonate  3  3  3  3  3  3   Titanium dioxide  5  5  5  5  5  5  Calculated nutrient concentration   CP  217  244  272  221  244  268   Crude fat  9  13  16  9  13  17   MJ ME (MJ/kg DM  15.5  15.3  15.2  15.3  15.3  15.3  1BD = basal diet; GD20 = genotype diet supplemented with 20% of 1 of 8 triticale or wheat genotypes; GD40 = genotype diet supplemented with 40% of 1 of 8 triticale or wheat genotypes. 2Mixture of the 8 triticale genotypes used in experiment 1. 3Mixture of the 8 wheat genotypes used in experiment 2. 4MCPh = monocalcium phosphate monohydrate. 5Vitamin premix (Raiffeisenkraftfutterwerke Süd GmbH, Würzburg, Germany) ingredients per kilogram of premix: vitamin A, 6,000,000 IU; vitamin D3, 1,500,000 IU; vitamin E, 15,000 mg; vitamin B1, 1,500 mg; vitamin B2, 3,000 mg; vitamin B6, 3,000 mg; vitamin B12, 15,000 μg; vitamin K3, 1200 mg; nicotinic acid, 25,000 mg; pantothenic acid, 7,000 mg; biotin, 50,000 μg; folic acid, 500 mg. 6Mineral premix (Gelamin SG 1, GFT mbH, Memmingen, Germany) provided per kilogram of complete diet: Mn, 120 mg; Fe, 90 mg; Zn, 80 mg; Cu, 15 mg; I, 1.6 mg; Co, 0.6 mg; Se, 0.5 mg. View Large Table 2. Analyzed phytase activity (FTU/kg DM) and total Ca and P (g/kg DM) of the diets used in broiler experiments 1 (triticale) and 2 (wheat) (g/kg DM of feed).1   BD  GD1  GD2  GD3  GD4  GD5  GD6  GD7  GD8  C (%)  102  20  40  20  40  20  40  20  40  20  40  20  40  20  40  20  40  Experiment 1   Phytase activity3  <100  210  640  480  890  360  850  360  610  360  660  210  550  270  550  230  690   Ca  3.91  4.86  5.71  4.92  5.67  4.89  5.70  4.90  5.71  4.84  5.66  5.04  5.96  4.73  5.96  5.10  5.82   P  2.64  3.35  4.12  3.38  4.28  3.58  4.21  3.45  4.15  3.59  4.22  3.48  4.33  3.37  4.13  3.25  4.05  Experiment 2   Phytase activity3  300  530  1270  660  1080  830  1710  770  1470  680  1160  810  1720  950  1590  510  1120   Ca  3.64  4.46  5.35  4.44  5.45  4.42  5.04  4.41  5.48  4.46  5.54  4.48  5.29  4.55  5.39  4.55  5.45   P  2.50  3.17  4.13  3.28  4.35  3.13  4.20  3.25  4.41  3.31  4.35  3.31  4.14  3.41  4.36  3.22  4.12    BD  GD1  GD2  GD3  GD4  GD5  GD6  GD7  GD8  C (%)  102  20  40  20  40  20  40  20  40  20  40  20  40  20  40  20  40  Experiment 1   Phytase activity3  <100  210  640  480  890  360  850  360  610  360  660  210  550  270  550  230  690   Ca  3.91  4.86  5.71  4.92  5.67  4.89  5.70  4.90  5.71  4.84  5.66  5.04  5.96  4.73  5.96  5.10  5.82   P  2.64  3.35  4.12  3.38  4.28  3.58  4.21  3.45  4.15  3.59  4.22  3.48  4.33  3.37  4.13  3.25  4.05  Experiment 2   Phytase activity3  300  530  1270  660  1080  830  1710  770  1470  680  1160  810  1720  950  1590  510  1120   Ca  3.64  4.46  5.35  4.44  5.45  4.42  5.04  4.41  5.48  4.46  5.54  4.48  5.29  4.55  5.39  4.55  5.45   P  2.50  3.17  4.13  3.28  4.35  3.13  4.20  3.25  4.41  3.31  4.35  3.31  4.14  3.41  4.36  3.22  4.12  1BD = basal diet; GD1–8 = genotype diets supplemented with 20% or 40% of 1 of 8 triticale or wheat genotypes. C = concentration of genotype. 2Mixture of the 8 genotypes of triticale or wheat used in experiment 1 and 2, respectively. 3Determined at pH 5 and 45°C, as described by Greiner and Egli (2003), U/kg. View Large Birds, Animal Management, and Sampling Procedure Two animal experiments were conducted to determine the pcdP of both grains. Experiment 1 investigated triticale and experiment 2 investigated wheat. Both experiments were carried out in the Agricultural Experiment Station of Hohenheim University, location Lindenhöfe, Eningen, Germany, in accordance with German Animal Welfare legislation. All procedures regarding animal handling and treatments were approved by the Animal Welfare Commissioner of the University. Broiler hatchlings (Ross 308, unsexed) were obtained from a local hatchery (Brüterei Süd GmbH and Company KG, Regenstauf, Germany). Birds were raised in floor pens (115 × 115 cm) on wood shavings and fed a commercial starter feed until they were 20 d old (0.90% Ca, 0.65% P, 21.5% CP, 6.3% ether extract, 12.6 MJ ME/kg, and 600 FTU/kg 3-phytase [EC 3.1.3.8, 4a E1600]). In both experiments, birds underwent routine vaccination against coccidiosis (via starter diets) and Newcastle disease on d 12, respectively. On d 20, birds were weighed and randomly allocated to 102 (experiment 1) or 106 floor pens (experiment 2), with 12 birds/pen. In experiment 1 each dietary treatment (BD and 16 triticale GD) was assigned to 6 pens (n = 6 replicates) according to a non-randomized complete block design. In experiment 2, 10 pens received BD treatment (n = 10 replicates), whereas for wheat GD 6 pens (n = 6 replicates) were used. Treatments were arranged in a modified α-design. The α-design is based on a design with 18 treatments tested in 6 complete replicates each with 2 blocks of size 9 within a replicate. Two of these treatments were associated with BD, the other 16 treatments were associated with wheat GDs. In the end, 2 out of 6 observations of the second BD treatment were randomly dropped. In both experiments, pens were arranged in an animal house with 4 columns of 22 to 28 pens per column and 6 sub-columns of 11 to 14 pens per column. Feed and tap water were offered for ad libitum consumption. Experimental diets were fed to animals for 5 or 6 d from d 20 onwards. The average daily feed intake (ADFI) and average daily gain (ADG) were recorded. During the first 2 d, the room temperature was set at 34°C, followed by a subsequent stepwise reduction of 0.5°C per d. Artificial lightening was provided at an intensity of 10 lx. During the first 2 d, light was provided for 24 h; thereafter, provision of light was reduced to 18 h per d. In experiment 1, birds from 4 out of 6 pens per treatment were sacrificed on d 25 of age, whereas samples from the remaining birds were taken on d 26. In experiment 2, all birds were sacrificed at 26 d of age. Animals were stunned with a mixture of 35% CO2, 35% N2, and 30% O2, and euthanized via CO2 asphyxiation. The abdominal cavity of each animal was immediately opened, the digestive tract removed, and the ileum (section between Meckel's diverticulum and 2 cm anterior to the ileo-ceco-colonic junction) dissected. The digesta of the distal half of the ileum was gently flushed out with double-distilled water (4°C) and pooled for all birds per pen into 1 sample per replicate. Samples were immediately frozen at –18°C, freeze-dried (Type Delta 1–24, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode, Germany), ground to pass through a 0.12-mm sieve screen, at a speed of 6,000 rpm (ZM 200 Ultra Centrifugal Mill, Retsch GmbH, Haan, Germany), and stored at 4°C until chemical analysis. Chemical Analyses The DM content of feed and digesta samples was analyzed according to the official methods used in Germany (Verband Deutscher Landwirtschaftlicher Untersuchungs- und Forschungsanstalten [VDLUFA] 1976); Method 3.1). Calcium, P, and Ti in feed and digesta samples were analyzed using an inductively coupled plasma optical emission spectrometer following a sulfuric and nitric acid wet digestion as described by Zeller et al. (2015a). Intrinsic phytase activity of experimental diets was analyzed by the direct incubation method (quantification of liberated inorganic P) at pH 5 and 45°C according to the method described by Greiner and Egli (2003). Calculations and Statistics The body weight (BW), ADG, ADFI, and feed-to-gain ratio were measured on a per-pen basis and adjusted for mortality, which was recorded daily. Prececal digestibility of P (y) from experimental diets was calculated on a pen basis according to the following equation:   \begin{eqnarray} y\left( \% \right) &=& 100 - 100{\rm{ }} \times \ \left( {\frac{{{\rm{Ti}}\,{\rm{in}}\,{\rm{the}}\,{\rm{diet(g/kg\ }}\,{\rm{DM)}}}}{{{\rm{Ti}}\,{\rm{in}}\,{\rm{the}}\,{\rm{digesta(g/kg}}\,{\rm{DM)}}}}} \right)\nonumber\\ && \times \left( {\frac{{{\rm{P}}\,{\rm{in}}\,{\rm{the}}\,{\rm{digesta(g/kg}}\,{\rm{DM)}}}}{{{\rm{P}}\,{\rm{in}}\,{\rm{the}}\,{\rm{diet(g/kg}}\,{\rm{DM)}}}}} \right) \end{eqnarray} (1)The amount of pcdP (mg/d) was calculated by multiplying the daily total P intake with diet (mg/d) and the respective digestibility (%), divided by 100. In 1 of the 6 pens receiving BD treatment, implausible performance and pcdP data (negative values, which differed twice the SD from means) were generated, and the results from this pen were not included in the data analysis. Statistical analyses were performed using the software package SAS for Windows (Version 9.3, SAS Institute, Cary, NC). Performance data and pcdP values of experimental diets were analyzed using a mixed model approach (procedure PROC MIXED) considering the treatment factors diet type (BD vs. GD), diet, and concentration of genotype as fixed factors and effects of block (just in experiment 2) and replicate as random effects. If the model fit was improved, additional random effects of “column” and “sub-column” was considered in the model. These factors were included via post-blocking according to the allocation of pens in the animal house. The model can be described by:   \begin{eqnarray} {y_{ijklmno}} &=& \mu + {\alpha _i} + {\beta _{ij}} + {\gamma _{ik}} + {\left( {\beta \gamma } \right)_{ijk}} + re{p_l} + {b_{lm}}\nonumber\\ && +\, {c_n} + {s_{no}}_{} + {e_{ijklmno}}, \end{eqnarray} (2)where μ = general mean; αi= effect of the ith diet type; βj = effect of the jth diet within diet type; γk = effect of the kth concentration of genotype within diet type; (βγ)jk = interaction effect of the jth diet and kth concentration of genotype within diet type; repl = effect of the lth replicate, blm = effect of the random mth block; cn = random effect of the nth column; sno = random effect of the oth sub-column within the nth column, and eijklmno = error of observation yijklmno. Block effects were only fitted for experiment 2. To ensure normal distribution and variance homogeneity of residuals, data on pcdP (%) were subjected to arcsine square-root transformation prior to analysis. Least-square means from the analysis were back-transformed for presentation only. A multiple t-test for treatment comparisons was applied only after a significant F-test. The level of significance was set at α = 0.05. Prececal digestibility of P from triticale genotypes was calculated using a mixed-model approach similar to that used to determine the prececal digestibility of amino acids from cereal grains, recently published by Zuber et al. (2016). This approach implies there is a linear relationship between P intake and digested P within the range of P intakes. The model used for triticale genotypes can be described as follows:   \begin{equation}{y_{ilmno}} = \mu + r \times {\beta _i} + re{p_l} + {b_{lm}} + {c_n} + {s_{no}} + {e_{ilmno}},\end{equation} (3)where: μ = intercept (representing the digestibility of the BD); r = ratio of the daily P intake attributable to the triticale genotype and the daily P intake attributable to the BD; βi = regression coefficient of genotype i; repl = effect of the lth replicate; blm = random effect of the mth block; cn = random effect of the nth column; sno = random effect of the oth sub-column, and eilmno = error of yilmno. The slope βi between r and yilmno(βi) represents the P digestibility of triticale genotype i. Regression coefficients were calculated simultaneously using the PROC MIXED procedure of SAS and compared between genotypes via contrasts using the ESTIMATE statement of the PROC MIXED procedure. Although diets were low in P, the assumption of a linear relationship between P intake and pcdP (mg/d) could not be confirmed in experiment 2. Thus, model (3) was adapted to fit separate slopes for both concentrations of all 8 wheat genotypes (in total 16 slopes) to estimate the pcdP (%). Relationships between pcdP and different chemical or physical characteristics of triticale and wheat genotypes as previously reported (Rodehutscord et al., 2016) were examined by regressing them to P digestibility values βi. The following regression was assumed:   \begin{equation} {\beta _i} = \pi + \tau \times {x_i} + {\varepsilon_i},\end{equation} (4)where π corresponds to a general digestibility, τ is the slope parameter between digestibility and chemical or physical property measure xi for ith genotype and εi is the random genotype specific deviation of digestibility of genotype i from the regression line. A significant test of τ corresponds to a relationship between digestibility and the corresponding property. To estimate τ, βi in (3) was replaced with (4) and the resulting model was re-run. Note that βi was multiplied with r in (3), thus each of the 3 effects in (4) is multiplied with r. As 92 properties were tested for relationship in both grains, we account for multiple testing by a Bonferroni correction of α. RESULTS Experiment 1 (triticale) As expected, the diet type (BD vs. GD) had a significant (P < 0.05) effect on the performance of birds and on the pcdP of the experimental diets (Table 3). Moreover, the concentration of triticale genotypes had a significant (P < 0.05) effect on performance data. The increased ADG (and decreased feed: gain ratio indicated that birds fed the GD performed better than those fed the BD. Additionally, the birds’ performance was improved by increasing the concentration of triticale genotypes in the diet from 20% to 40%. Table 3. BW1, ADG1, ADFI1, P intake1, feed-to-gain ratio1 and pcdP of broilers fed basal diet (BD) or diets containing different concentrations (C) of triticale genotypes (GD1–8) in experiment 1.2 Effect  BW (g)  ADG (g/d)  ADFI (g/d)  Feed:gain (g/g)  P intake (mg/d)  pcdP (mg/d)  pcdP (%)  Diet type3   BD  942  8  59  9.00  142  53  36.5   GD  1030  23  79  3.77  276  146  52.7   SEM  18.3  1.7  2.5  0.551  8.6  8.7  2.90   P value  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  Diet (Diet type)   GD1  1024  19  77  4.85  264  133  51.1   GD2  1025  22  82  3.91  287  153  53.5   GD3  1022  24  78  3.50  278  150  53.4   GD4  1027  23  76  3.64  265  126  46.7   GD5  1048  25  82  3.42  294  161  55.0   GD6  1010  23  77  3.64  276  151  54.7   GD7  1043  25  79  3.50  270  146  54.2   GD8  1042  23  81  3.67  271  145  52.8   SEM  17.1  1.57  2.3  0.503  8.0  7.9  2.67   P value  0.423  0.233  0.099  0.540  0.008  0.022  0.389  C (Diet type)   GD20  1012  19  75  4.33  236  122  51.7   GD40  1049  27  83  3.20  315  170  53.6   SEM  13.1  0.8  1.7  0.252  6.0  4.9  1.40   P value  <0.001  <0.001  <0.001  0.002  <0.001  <0.001  0.312  Diet × C (Diet type)   P value  0.228  0.475  0.617  0.810  0.562  0.509  0.506  Effect  BW (g)  ADG (g/d)  ADFI (g/d)  Feed:gain (g/g)  P intake (mg/d)  pcdP (mg/d)  pcdP (%)  Diet type3   BD  942  8  59  9.00  142  53  36.5   GD  1030  23  79  3.77  276  146  52.7   SEM  18.3  1.7  2.5  0.551  8.6  8.7  2.90   P value  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  Diet (Diet type)   GD1  1024  19  77  4.85  264  133  51.1   GD2  1025  22  82  3.91  287  153  53.5   GD3  1022  24  78  3.50  278  150  53.4   GD4  1027  23  76  3.64  265  126  46.7   GD5  1048  25  82  3.42  294  161  55.0   GD6  1010  23  77  3.64  276  151  54.7   GD7  1043  25  79  3.50  270  146  54.2   GD8  1042  23  81  3.67  271  145  52.8   SEM  17.1  1.57  2.3  0.503  8.0  7.9  2.67   P value  0.423  0.233  0.099  0.540  0.008  0.022  0.389  C (Diet type)   GD20  1012  19  75  4.33  236  122  51.7   GD40  1049  27  83  3.20  315  170  53.6   SEM  13.1  0.8  1.7  0.252  6.0  4.9  1.40   P value  <0.001  <0.001  <0.001  0.002  <0.001  <0.001  0.312  Diet × C (Diet type)   P value  0.228  0.475  0.617  0.810  0.562  0.509  0.506  1Average of birds slaughtered 25 and 26 d post hatch. 2Data are given as LS means. BD had n = 5 and GD1–8 had n = 6 pens per treatment with 10 birds per pen (GD320 and GD640: BW gain and F: G had n = 5). 3Diet type = BD vs. GD. View Large The pcdP of the BD was on average (standard error [SE] in parentheses) 36% (4.0%), and was 17% lower than that of the GD (54% [3.7%]). Neither the concentration of genotypes nor the diet itself affected the pcdP (%) of GD. However, P intake and the pcdP (mg/d) significantly (P < 0.05) differed between diets, with highest values found for GD2 and GD5 and the lowest for GD1 and GD4. No interaction was found between diet and concentration of triticale genotypes on the performance of birds or on the pcdP of experimental diets. The pcdP of triticale genotypes estimated by linear regression according to model 3 varied between triticale genotypes and ranged from 53% to 78% (Table 4). The pcdP for genotype no. 4 (53%) was significantly (P < 0.05) lower compared with genotype no. 3, 5, 6, and 8 (75%, 74%, 73%, 78%). A significant correlation was not observed between the chemical and physical properties of genotypes and their pcdP. Table 4. Estimated digestibility (%) and SE of the triticale genotypes fed to broilers in experiment 1.1   Triticale genotype2      1  2  3  4  5  6  7  8  P value3  Estimate  63.7a,b  72.0a,b  74.6a  52.9b  74.2a  73.2a  71.1a,b  78.2a                      <0.001  SE  13.40  9.40  9.39  9.97  9.32  9.02  10.25  10.98      Triticale genotype2      1  2  3  4  5  6  7  8  P value3  Estimate  63.7a,b  72.0a,b  74.6a  52.9b  74.2a  73.2a  71.1a,b  78.2a                      <0.001  SE  13.40  9.40  9.39  9.97  9.32  9.02  10.25  10.98    1Genotypes 1–8 had n = 6 pens per treatment with 10 birds per pen. 2The slopes (βi) between r and yijkl from equation 3 multiplied by 100 represent the digestibility of the respective triticale genotype i. 3Estimates within a row not sharing a common superscript differ significantly (multiple t-tests in case of interaction), P ≤ 0.05. View Large Experiment 2 (wheat) Similar to experiment 1, diet type had a significant (P < 0.05) effect on the birds’ performance and on the pcdP of diets in experiment 2. In addition, similar to experiment 1, feeding of GD increased the BW of birds at d 26 of age, ADFI and ADG, and decreased the feed: gain ratio compared with BD (Table 5). The P intake and amount of pcdP (mg/d) also increased in animals fed the GD instead of the BD; however, in contrast to experiment 1, the average (SE) pcdP (%) decreased from 63% (1.5%) in the BD to 55% (1.83%) in the GD. Table 5. BW at d 26 of age, ADG, ADFI, P intake, feed-to-gain ratio, and pcdP of broilers fed basal diet (BD) or diets containing different concentrations (C) of wheat genotypes (GD1–8) in experiment 2.1 Diet  BW (g)  ADG (g/d)  ADFI (g/d)  Feed:gain (g/g)  P intake (mg/d)  pcdP (mg/d)2  pcdP (%)  BD  987  19  83  5.17  188  118  63.1  GD120  1039  28  100  3.64  287  180  62.8  GD140  1027  25  101  4.26  374  196  52.9  GD220  1025  27  96  3.62  285  173b  60.7  GD240  1004  24  96  4.10  374  195a  52.2  GD320  1026  25  93  3.78  262  154b  58.4  GD340  1014  21  104  4.97  391  201a  51.7  GD420  1018  28  103  3.72  301  173b  57.7  GD440  1037  30  106  3.70  417  210a  50.1  GD520  1046  31  100  3.31  297  192  64.7  GD540  1026  24  99  4.29  385  185  48.0  GD620  1052  29  108  3.71  323  191  59.0  GD640  1018  24  100  4.24  375  171  46.0  GD720  1028  27  102  3.81  313  193b  62.0  GD740  1055  30  105  3.59  413  217a  52.4  GD820  1024  27  98  3.91  286  168  58.4  GD840  1025  24  100  4.67  370  177  48.0  Pooled SEM2  17.2 (13.7)  2.1 (1.7)  3.8 (3.0)  0.413 (0.320)  11.9 (9.5)  8.4 (6.9)  1.83 (1.46)  ANOVA P value3  Diet type4  0.003  <0.001  <0.001  0.005  <0.001  <0.001  <0.001  Diet (Diet type)  0.778  0.204  0.212  0.633  0.004  0.003  0.007  C (Diet type)  0.440  0.022  0.431  0.011  <0.001  <0.001  <0.001  Diet × C (Diet type)  0.602  0.327  0.375  0.711  0.054  0.001  0.088  Diet  BW (g)  ADG (g/d)  ADFI (g/d)  Feed:gain (g/g)  P intake (mg/d)  pcdP (mg/d)2  pcdP (%)  BD  987  19  83  5.17  188  118  63.1  GD120  1039  28  100  3.64  287  180  62.8  GD140  1027  25  101  4.26  374  196  52.9  GD220  1025  27  96  3.62  285  173b  60.7  GD240  1004  24  96  4.10  374  195a  52.2  GD320  1026  25  93  3.78  262  154b  58.4  GD340  1014  21  104  4.97  391  201a  51.7  GD420  1018  28  103  3.72  301  173b  57.7  GD440  1037  30  106  3.70  417  210a  50.1  GD520  1046  31  100  3.31  297  192  64.7  GD540  1026  24  99  4.29  385  185  48.0  GD620  1052  29  108  3.71  323  191  59.0  GD640  1018  24  100  4.24  375  171  46.0  GD720  1028  27  102  3.81  313  193b  62.0  GD740  1055  30  105  3.59  413  217a  52.4  GD820  1024  27  98  3.91  286  168  58.4  GD840  1025  24  100  4.67  370  177  48.0  Pooled SEM2  17.2 (13.7)  2.1 (1.7)  3.8 (3.0)  0.413 (0.320)  11.9 (9.5)  8.4 (6.9)  1.83 (1.46)  ANOVA P value3  Diet type4  0.003  <0.001  <0.001  0.005  <0.001  <0.001  <0.001  Diet (Diet type)  0.778  0.204  0.212  0.633  0.004  0.003  0.007  C (Diet type)  0.440  0.022  0.431  0.011  <0.001  <0.001  <0.001  Diet × C (Diet type)  0.602  0.327  0.375  0.711  0.054  0.001  0.088  1Data are given as LS means. BD had n = 10 and GD1–8 had n = 6 pens per treatment with 12 birds per pen. 2Pooled SEM of GD (SEM of BD). 3Estimates within a GD having different superscripts differ significantly among the 2 concentrations of genotypes tested (multiple t-tests in case of interaction), P ≤ 0.05; due to the large number of possible comparisons we refer only to those differences between concentrations of wheat within a genotype. 4Diet type = BD vs. GD. View Large Increasing the concentration of wheat from 20% to 40% had a negative effect (P < 0.05) on the ADG and feed: gain ratio. Animals fed the GD40 showed a lower ADG (25 vs. 28 g/d) and a higher feed: gain ratio (4.2 vs. 3.7 g/g) than those fed the GD20. A higher concentration of wheat genotypes had a negative effect (P < 0.05) on the pcdP (%) of diets, which decreased from 60% (1.0%) in GD20 to 50% (1.0%) in GD40. Moreover, a significant effect of diet was observed on the pcdP (%). While GD1 and GD7 showed the highest pcdP (58% and 57%), significantly (P < 0.05) lower values were found for GD6 and GD8 (53%). In addition, the former diets also showed the highest ADG and lowest feed: gain ratio, but the lowest ADG and highest feed: gain ratio was observed for GD3. An interaction between diet and wheat genotype concentration was detected only for pcdP (mg/d). Thus, the results showed a significant (P < 0.05) increase in the amount of pcdP with the higher wheat genotype concentration (40%) in GD2, GD3, GD4, and GD7, whereas no differences between concentrations were found for the other GD. Estimates of the pcdP of wheat genotypes obtained by regression analysis are given in Table 6. The values ranged from 38% to 67% with inclusion of 20% wheat genotypes and from 20% to 38% with inclusion of 40% wheat genotypes. At the concentration of 20%, genotypes no. 1 and 5 showed the highest pcdP, which was significantly (P < 0.05) different from that of genotypes no. 3, 4, 6, and 8. At the concentration of 40%, the pcdP was highest for genotypes no. 1, 2, and 7, differing significantly (P < 0.05) from genotype no. 6, which showed the lowest pcdP. A significant (P < 0.05) decrease in the pcdP with increased wheat concentration was observed for all genotypes except for no. 3, 4, and 8. As for triticale genotypes, significant correlations were not observed between the chemical and physical properties of genotypes and their pcdP. Table 6. Estimated digestibility (%) and SE at different inclusion concentrations (C, %) of wheat genotypes contained in diets fed to broilers in experiment 2.1     Wheat genotype2      C  1  2  3  4  5  6  7  8  P value3  Estimate  20  59.4A,a,b  51.5A,bd  38.1d  39.5d  66.9A,a  43.4A,c,d  56.3A,ac  38.7d    SE    5.60  4.97  5.40  4.80  5.00  5.60  4.90  5.70                        <0.001  Estimate  40  36.7B,a  36.7B,a  34.7a,b  32.1a,b  28.5B,a,b  20.2B,b  38.0B,a  23.8a,b    SE    7.10  6.00  7.20  6.22  5.70  6.00  5.60  6.30        Wheat genotype2      C  1  2  3  4  5  6  7  8  P value3  Estimate  20  59.4A,a,b  51.5A,bd  38.1d  39.5d  66.9A,a  43.4A,c,d  56.3A,ac  38.7d    SE    5.60  4.97  5.40  4.80  5.00  5.60  4.90  5.70                        <0.001  Estimate  40  36.7B,a  36.7B,a  34.7a,b  32.1a,b  28.5B,a,b  20.2B,b  38.0B,a  23.8a,b    SE    7.10  6.00  7.20  6.22  5.70  6.00  5.60  6.30    1Genotypes 1–8 had n = 6 pens per treatment with 12 birds per pen. 2The slopes (βi) between r and yijkl from equation 3 multiplied by 100 represent the digestibility of the respective wheat genotype i at concentration of 20% or 40%. 3Estimates within a row not sharing a common lower case or within a column not sharing a common capital letter differ significantly (multiple t-tests in case of interaction), P ≤ 0.05. View Large DISCUSSION A standard trial protocol for determination of digestible P from different P sources is an important prerequisite for the harmonization of P evaluation and requirement standards for poultry (WPSA, 2013). In a future table of digestible P in feedstuffs data from the present study can contribute information on triticale and wheat. Previous studies based on the WPSA protocol have considered rapeseed meal (Olukosi et al., 2015), wheat distillers’ grains with solubles (Adebiyi and Olukosi, 2015), soybean meal (Rodehutscord et al., 2017), corn and canola meal (Mutucumarana et al., 2014b), and corn and wheat (Kupcikova et al., 2017). Effect of Triticale Genotype and its Concentration on Prececal P Digestibility The results of our experiments confirmed the hypothesis that the triticale or wheat genotype affects the pcdP of grains in broiler chickens. However, compared with wheat, the pcdP of triticale genotypes was less variable, ranging between 53% and 78%, with most of the triticale genotypes showing a value higher than 70%. Thus, most genotypes did not differ in their pcdP. This explains the missing effect of diet on the pcdP in GD, in which a maximum of one-third of total P comes from the test grain. These results are consistent with those of previous studies on broiler chickens fed diets containing 45% of 4 different triticale varieties, in which the variety had no effect on the ash content of tibiotarsi (Jondreville et al., 2007). The average (SE) pcdP of GD with triticale was 54% (3.7%) in the present study. This value is consistent with the results of Jamroz et al. (1996) and Jamroz et al. (1998), who detected a P retention of 46% and 51% from diets containing 55% or 40% triticale, respectively, in 6-wk-old broilers. No correlations between the pcdP and chemical or physical properties of triticale genotypes were detected in the present study. Jondreville et al. (2007) described a positive response of bone ash contents to intrinsic phytase of triticale in broiler chickens. However, consistent with our findings, those authors were unable to detect differences between diets with phytase activity of 620 to 1,390 U/kg mainly derived from triticale varieties with activities of 768 to 1,700 U/kg of grain. Moreover, our results revealed no effect of diet extract viscosity on the pcdP of triticale genotypes, although mineral absorption in broiler chickens from wheat- (van der Klis et al., 1995) or corn-based diets (Mohanna et al., 1999) has been shown to be impaired by increased diet viscosity. The extract viscosity of triticale genotypes investigated in the present study ranged only between 1.10 and 1.44 mPa·s (at shear rate of 380/s; Rodehutscord et al., 2016), which probably represents the variation being too low to allow any relationship between viscosity and pcdP of genotypes to be detected. In broiler chickens fed P-deficient diets based on different triticale varieties with viscosities of 1.79 to 2.7 mL/g, there was no difference in bone ash contents and variety did not affect animal performance (Jondreville et al., 2007). The latter was also observed in the present study and by Elangovan et al. (2011) with non P-deficient diets. While the increased amount of pcdP (mg/d) with higher P contents in triticale genotypes did not affect animal performance, the increased amount of pcdP (mg/d) with GD40 compared to GD20 was coincided with a higher BW, ADG, ADFI and feed to gain ratio. However, the positive effect of higher triticale concentrations on animal performance may be not only a result of increased pcdP (mg/d) as overall nutrient composition of GD40 (Table 1) changed with substitution of corn starch by further triticale. Effect of Wheat Genotype and its Concentration on Prececal P Digestibility For wheat-based diets, the pcdP of the GD ranged between 53% and 58%. These results are consistent with those of previous studies in broiler in which for diets based on different wheat varieties, P retention of 45% to 64% (Barrier-Guillot et al., 1996) and a pcdP of 47% to 55% (van der Klis et al., 1995) were reported. However, the differences found in the pcdP (%) or the amount of pcdP (mg/d) in the GD1–8 diets, did not affect animal performance in the present study. Similar to triticale, the concentration of wheat was more important with respect to animal performance than the diet itself. However, in contrast to experiment 1, the increase in wheat from 20% to 40% considerably decreased the ADG and increased the feed: gain ratio, although the amount of pcdP (mg/d) was either similar or increased with 40% wheat. Thus, factors other than the digestible P must have been of higher relevance for animal performance under the given experimental conditions. In the present study, a 10% decrease in the pcdP was observed with increased wheat inclusion; however, Mutucumarana et al. (2014a) found no effect of wheat concentration on the pcdP in 4-wk-old broiler chickens fed diets containing 24% to 95% wheat. Moreover, these authors reported a positive effect of increasing wheat proportions on animal performance. However, the semi-synthetic diets used by Mutucumarana et al. (2014a) contained very low P contents at 0.85 to 3.08 g total P/kg, and showed a pcdP of only 42%. Thus, animal performance was even lower than that reported in the present study, which may explain the different response of animals to wheat concentration. The pcdP of the test wheat in the experiment of Mutucumarana et al. (2014a) was calculated to be 46%. From diets based on 99% (Wu et al., 2004) or 94% (Rutherfurd et al., 2002) wheat, a pcdP of 51% or 50% was reported for 5-wk-old broiler chickens, respectively. These values are in the range of the pcdP determined for wheat genotypes at the concentration of 20% in the present study. However, our results indicated that pcdP was reduced by 3% to 38% when the wheat concentration was increased from 20% to 40%. In contrast, Mutucumarana et al. (2014a) described a linear relationship between dietary P content and P output in digesta samples in the range of 24% to 95% of dietary wheat. In their study, the within-treatment variation considerably increased with wheat concentrations higher than 24%. As a result, linear regression analysis for the wheat tested by Mutucumarana et al. (2014a) showed a reduced goodness-of-fit than has been reported for other P sources, such as corn, canola meal (Mutucumarana et al., 2014a), and soybean meal (Rodehutscord et al., 2017). Thus, it remains unclear whether the amount of pcdP from wheat linearly increases with increasing dietary P derived from wheat in growing broiler chickens. Our data, which were obtained from several wheat genotypes and were based on more observations than used by Mutucumarana et al. (2014a), do not confirm linearity. A recent digestibility ring test revealed great differences in the results between institutions that determined the pcdP of soybean meal (Rodehutscord et al., 2017). The authors hypothesized factors like feeding and housing in the pre-experimental period might influence the degradation of phytate P and pcdP. Thus, comparisons between P digestibility studies, even if conducted under similar experimental conditions, should be made with great caution until the WPSA protocol is more refined. In the present study, correlation analysis revealed no relationship between total or phytate P contents in wheat genotypes and the pcdP of test wheats. These results are in accordance with those of former studies (Barrier-Guillot et al., 1996), in which total and phytate P contents of different wheat varieties were not related to the P retention of broilers from wheat-based diets. Additionally, no correlation was detected between the intrinsic phytase activity of wheat genotypes and the pcdP. In contrast, Barrier-Guillot et al. (1996) reported a positive correlation between the phytase activity of wheat varieties and P retention of broilers. However, when considering from their data only the wheat varieties that had been grown under similar agronomic conditions, no correlation could be found between phytase activity and P retention. Moreover, a minor effect of the intrinsic phytase activity in wheat on broiler chickens’ pcdP was suggested by Shastak et al. (2014), Zeller et al. (2015b), and Leytem et al. (2008a, b). Additionally, no effect of the extract viscosity of wheat genotypes was observed on the pcdP in the present study. In contrast, van der Klis et al. (1995) reported a decreased pcdP from corn-wheat-based diets with increasing extract viscosity of the different wheat varieties fed to broiler chickens. However, although supplementation of xylanase decreased the viscosity of the jejunal and ileal digesta in the studies of van der Klis et al. (1995), the enzyme did not increase the pcdP from diets. Thus, factors other than digesta viscosity must have been more important for the P digestibility. Zuber and Rodehutscord (2016) determined the digestibility of amino acids in laying hens from 20 different wheat genotypes, including the 8 genotypes used in the present study. As found in our study, neither physical nor chemical properties sufficiently explained the differences observed between genotypes. These authors hypothesized that differences in the fine structure, substitution pattern, or structural arrangement of NSP may affect solubility and other properties of polymers in the gastrointestinal tract, which in turn, can influence the digestibility of nutrients, but cannot be described by the method used for carbohydrate analysis of genotypes in the present study. To conclude, the results of the present study confirm our hypothesis that P digestibility from triticale and wheat in broiler chickens is influenced by genotype. The physical and chemical properties used for characterization of triticale and wheat genotypes did not explain the differences found in P digestibility. 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Poultry Science – Oxford University Press

Published: Mar 1, 2018

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