Relationship between total crude protein content and apparent ileal amino acid digestibility of Australian wheat for broilers D. ZHANG, X. LI and W. L. BRYDEN School of Animal Studies, University of Queensland, Gatton, QLD 4343, Australia *Corresponding author: d.zhang@uq.edu.au Wheat is the most common ingredient in Australian poultry diets and may contribute most of the energy and a great portion of amino acids in the diet. Small differences in nutrient content or digestibility can affect the bird performance. Theoretically the most logical way to assess protein quality is by consideration of amino acid availability of the ingredients. The availability of amino acids is very difficult to determine, but digestibility is a good estimate of availability. Twenty one locally grown wheat samples were obtained from commercial sources and tested for total and digestible crude protein and amino acid contents. In this study apparent ileal amino acid digestibility was determined in broilers. The relationship between total crude protein and amino acid contents, and total crude protein and ileal digestible amino acid contents were examined. The results of the study show that the concentrations of individual amino acids in wheat are influenced by grain crude protein content. Total and digestible amino acid concentrations in wheat increased linearly with increasing levels of total crude protein. The highly significant positive correlations indicate that it is possible to predict the total and digestible amino acid contents from total crude protein content of wheat samples. Keywords: Wheat; protein; amino acids; digestibility; broilers. Introduction Wheat is the most popular species among the cereals and the most common cereal ingredient in Australian poultry diets. As wheat may contribute most of the energy and a great portion of amino acids in the diet, small differences in nutrient content or digestibility of wheat can affect bird performance. Considerable research has been reported on the apparent metabolisable energy (AME) values of Australian wheats and the values have been found to vary widely from 11.9 to 15.3 MJ/kg DM (Mollah et al., 1983; Black et al., 2005). However, data on the amino acid digestibility of Australian wheats are limited as amino acid analysis is too costly and time consuming for routine investigation in the feed industry. It would be useful if amino acid content could be predicted from its crude protein content which is much more readily determined. Digestible amino acids values of the ingredients are preferred in the compilation of mixed poultry diets as Bryden and his colleagues (see Ravindran and Bryden, 1999a,b; Li et al., 2002) have shown that formulating diet based on digestible amino acid values are superior to total amino acid values in term of bird growth performance. The beneficial effects of using digestible amino acid values in broiler diet formulations were also reported by others (Fernadez et al., 1995; Rostagno et al., 1995; Douglas and Parsons, 1999). The objective of the present study was (1) to determine the ileal amino acid digestibility of locally grown wheats, and (2) to examine the relationships between total crude protein and amino acid contents, and total crude protein and ileal digestible amino acid contents. 1
Material and methods Experimental procedures Male broiler chickens that had been fed commercial broiler diets were individually weighed at day 34 post-hatching and the birds with body weights closest to the mean were selected and randomly allocated into pens (7 birds/pen). Fifty kilogram samples of 21 locally grown wheats were obtained from commercial sources. The diet for the amino acid digestibility assay contained 918 g wheat/kg as the only source of protein. All diets contained per kilogram 19 g dicalcium phosphate and 10 g limestone. No attempt was made to balance the dietary concentrations of calcium and phosphorus. Vitamin-mineral Premix was added as manufacturer recommended at 2.5 kg/tonne. Celite (20 g/kg) was added to all diets as a source of acidinsoluble ash (AIA) which was used as an indigestible marker. Diets were provided ad libitum and water was available at all times. Each assay diet was fed in a mash form to three pens from 35 to 42 days of age. On day 42, all birds were euthanased by an intracardial injection of sodium pentobarbitone solution. The contents of the lower half of the ileum were collected by gently flushing with distilled water into plastic containers. Ileal digesta from birds within a pen were pooled and immediately frozen at -20 C. Samples were subsequently freeze-dried and ground to pass through a 0.5 mm sieve prior to chemical analysis. All experimental procedures were approved by the University of Sydney Animal Care and Ethics Committee and complied with the Australia Code of Practice for the Care and Use of Animals for Scientific Purposes. Chemical analysis The total nitrogen content of ingredient and ileal digesta samples (approximately 0.5 g) was determined by the Dumas combustion method using a Leco (LECO Corporation, St. Joseph, Michigan, USA) FP-428 nitrogen analyser (AOAC, 1998). The value for nitrogen was multiplied by 6.25 to obtain the total crude protein content of the sample. Acid insoluble ash was determined using the method of Mollah et al. (1983). Briefly 1-2 g of samples were weighed into sintered-glass crucibles (Pyrex) and dried for 24 hr at 105 C in a drying oven. The crucibles were then cooled in a desiccator and weighed. The dried samples were ashed at 500 C for 8 hr. After ashing, crucibles were placed in a crystallising dish and boiled with 4 M hydrochloric acid. Hydrochloric acid was removed under suction and the residue was rinsed with purified water. The procedure was repeated until the sample appeared white. The crucibles were then oven-dried (105 C) and re-weighed. Total amino acid contents were determined on a Shimadzu LC-10A amino acid analyser (Shimadzu Corp, Kyoto, Tokyo, Japan) from hydrolysates obtained by hydrolysis of ingredient and ileal digesta samples containing 80 mg crude protein with 40 ml of 8 M hydrochloric acid in 50 ml Schott bottle under pressure of 100 kpa at 121 o C for 16 hrs. Amino acids in the hydrolysate were separated using cation-exchange column chromatography with post-column derivatisation and fluorometric detection with O-phthaldialdehyde (Li et al., 2006). Apparent ileal crude protein and amino acid digestibility coefficients were calculated using AIA as a marker as fellows: (AA/AIA)d (AA/AIA)i Apparent ileal amino acid digestibility coefficients = (AA/AIA)d Where, (AA/AIA)d = ratio of amino acid to acid insoluble ash in diet and (AA/AIA)i = ratio of amino acid to acid insoluble ash in ileal digesta. The calculations were based on the assumption that the marker was 100% recoverable in excreta. 2
Statistical analysis All the data were analysed according to the method of Steel et al. (1997) using the Minitab program version 11.0 (Minitab, 1996). Linear regression analysis was used to determine the relationship between different parameters. The level of significance was set at P < 0.05. Results and discussion The total crude protein content of wheat samples varied from 93 g/kg to 172 g/kg. The concentrations of total amino acids were in the ranges which were reported by Ravindran et al. (1998). With increasing protein levels, the concentrations of all amino acids increased significantly (R 0.896, P < 0.001) (Table 1). The present results show that the concentrations of individual amino acids in wheat are influenced by grain protein content. The highly significant positive correlations indicate that it is possible to predict the amino acid contents from the crude protein content of wheat samples. It may be argued that increases in grain protein content increased the concentrations of individual amino acids but do not reflect corresponding improvements in nutritive value because the increases are mainly achieved by increases in gluten (the storage protein fraction) content. The correlation coefficient values between total crude protein and total amino acid concentration in wheat protein were negative for most amino acids, the exception being glutamic acid. With increasing protein levels, the concentrations of aspartic acid, threonine, glycine, alanine, valine, leucine, lysine and arginine in wheat grain protein decreased (P<0.05 to 0.001). In contrast, concentration of glutamic acid in the protein increased (P<0.01) with increasing protein contents. Negative correlations between the protein content and concentration of most essential amino acids in wheat protein indicate that the nutritive value is lowered with increasing protein contents. However the slightly lower content of the first three nutritional limiting amino acids (lysine, methionine and threonine) in high protein wheat is more than offset by the greater total amount of limiting amino acids due to the higher percentage of protein in the wheat. Table 1 Intercept, slope and correlation coefficients of regression lines describing total amino acid (g/kg air dry basis) content as a function of total crude protein (g/kg air dry basis) content in wheat. Amino acid Intercept (a) Slope (b) Correlation Coefficient P value Aspartic acid 1.41 0.039 0.962 <0.001 Threonine 0.553 0.027 0.963 <0.001 Serine 0.108 0.057 0.983 <0.001 Glutamic acid -7.325 0.371 0.985 <0.001 Glycine 0.651 0.036 0.974 <0.001 Alanine 1.404 0.025 0.989 <0.001 Valine 0.632 0.038 0.980 <0.001 Methionine -0.006 0.012 0.896 <0.001 Isoleucine -0.196 0.038 0.980 <0.001 Leucine 0.523 0.064 0.996 <0.001 Tyrosine -0.566 0.031 0.928 <0.001 Phenylalanine -0.372 0.049 0.966 <0.001 Histidine 0.220 0.024 0.970 <0.001 Lysine 1.729 0.016 0.901 <0.001 Arginine 1.023 0.038 0.966 <0.001 Y = a +bx Y = Total amino acid content (g/kg air dry basis) X = Total crude protein content (g/kg air dry basis) The average ileal digestibility of amino acids in different wheats varied from 0.70 to 0.84. The differences in digestibility between wheat samples were approximately 13 percentage units for crude protein and 16 percentage units for lysine. There were significant positive correlations between total crude protein content and digestible amino acid content in wheat (R>0.91, P<0.01) (Table 2). The amino acids in higher protein wheat were more digestible than those in lower protein wheat. Similarly significant positive correlations (R> 3
0.91, P<0.001) between digestible crude protein and digestible amino acid concentrations have been found in wheat. The protein effect was particularly marked for lysine and threonine, the two amino acids which are most likely to be deficient when chickens are fed wheat-based diets. These two amino acids were shown to be the least digestible in wheat. The digestibility of lysine varied among wheat samples and was generally lower than other essential amino acids (except for threonine). However, digestibility of lysine tended to be higher in high protein cultivars compared to low protein cultivars. Although almost 70% of wheat proteins are present in the starchy endosperm, lysine is mainly found in the aleurone layer (Biely, 1973). Wheat proteins located in the endosperm would be expected to be easier to digest than those in the aleurone layer. Table 2. Intercept, slope and correlation coefficients of regression lines describing ileal digestible amino acid content (g/kg air dry basis) as a function of total crude protein content (g/kg air dry basis) in wheat grain. Digestible N Intercept (a) Slope (b) Correlation Coefficient P value Crude protein 21-15.936 0.941 0.996 <0.001 Aspartic acid 21 0.211 0.036 0.952 <0.001 Threonine 21-0.274 0.025 0.940 <0.001 Serine 21-0.886 0.055 0.970 <0.001 Glutamic acid 21-8.656 0.361 0.983 <0.001 Glycine 21-0.160 0.033 0.964 <0.001 Alanine 21 0.585 0.023 0.977 <0.001 Valine 21-0.144 0.036 0.978 <0.001 Methionine 21-0.095 0.011 0.915 <0.001 Isoleucine 21-0.694 0.036 0.981 <0.001 Leucine 21-0.426 0.062 0.991 <0.001 Tyrosine 21-1.034 0.029 0.915 <0.001 Phenylalanine 21-1.047 0.049 0.966 <0.001 Histidine 21-0.236 0.022 0.974 <0.001 Lysine 21 0.919 0.015 0.911 <0.001 Arginine 21 0.330 0.034 0.952 <0.001 Y = a +bx Y = Ileal digestible amino acid contents (g/kg air dry basis) X = Total crude protein content (g/kg air dry basis) In conclusion, the present results indicate that ileal amino acid digestibility of high protein wheat cultivars are greater than those of lower protein cultivars. The amino acid digestibility in wheat therefore appears to be largely influenced by crude protein content. The highly significant positive correlations indicate that it is possible to predict the total and digestible amino acid contents from total crude protein content of wheat samples. References AOAC. (1998) Official Methods of Analysis, 16 th edn. Association of Official Analytical Chemists, Washington, DC. Biely, J. (1973). The Nutritive value of wheat In The Nutritive Value of Wheat: In Poultry Rations. Published by the Canadian Wheat Board. pp. 5-28. BLACK, J.L., HUGHES, R.J., NIELSEN, S.G., TRENREA, A.M., MACALPINE and VAN BARNEVELD, R.J. (2005). Aust. Poult. Sci. Symp., 17: 21-29 DOUGLAS, M.W. and PARSONS, C.M. (1999). Dietary formulation with rendered spent hens meals on a total amino acids versus a digestible amino acids basis. Poult. Sci., 78:556-560. FERNANDEZ, S.R., ZHANG, Y. and PARSONS, C.M. (1995). Dietary formulation with cottonseed meal based on total amino acid versus digestible amino acid basis. Poult. Sci., 74: 1168-1179. LI, X., HIGGINS, T. J.V. and BRYDEN, W.L. (2006). Biological response of broiler chickens fed peas (Pisum sativum L.) expressing the bean (Phaseolus vulgaris L.) α-amylase inhibitor transgene. J. Sci. Food Agric. In press. 4
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