Utilisation of digestible amino acids by broilers

Transcription

1 Utilisation of digestible amino acids by broilers A report for the Rural Industries Research and Development Corporation By W.L. Bryden and X. Li February 2004 RIRDC Publication No 04/030 RIRDC Project No US-80A

2 2004 Rural Industries Research and Development Corporation and University of Sydney. All rights reserved. ISBN X ISSN Improving the utilisation of dietary amino acids in meat chickens Publication No. 04/030 Project No. US-80A The views expressed and the conclusions reached in this publication are those of the author and not necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person who relies in whole or in part on the contents of this report. This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone Researcher Contact Details Professor W.L. Bryden School of Animal Studies University of Queensland Gatton Qld 4343 Phone: Fax: hossas@uqg.uq.edu.au In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: Fax: rirdc@rirdc.gov.au. Website: Published in February 2004 Printed on environmentally friendly paper by Canprint ii

3 Foreword The supply of amino acids in the form of protein constitutes one of the most costly components of poultry diets. Therefore, it is essential to optimise the utilisation of these nutrients. Our understanding of amino acid availability is a major avenue for improving optimisation. Currently, digestibilities of amino acids determined at the terminal ileum are considered to give good estimates of amino acid availability. However, few commercial nutritionists formulate diets based solely on digestible amino acid because of wide variations in digestible amino acid values published from different sources, arising from difference in sample variation, type of birds, assay diets and assay methodology. This publication details a series of studies which examined the influence of a number of factors on the observed variability in amino acid digestibility. Age of bird, feed enzymes and feed ingredient processing were all shown to influence ileal amino acid digestibility of feedstuffs. Broilers fed diets formulated on a digestible amino acid basis had superior growth performance and breast meat yield compared to birds fed diets formulated on a total amino acid basis. This project was funded by industry revenue that is matched by funds provided by the Australian Government. This report, a new addition to RIRDC s diverse range of over 1000 research publications, forms part of our Chicken Meat R & D Program, which aims to support increased sustainability and profitability in the chicken meat industry by focussing research and development on those areas that will enable the industry to become more efficient and globally competitive and that will assist in the development of good industry and product images. Most of our publications are available for viewing, downloading or purchasing online through our website: downloads at purchases at Simon Hearn Managing Director Rural Industries Research and Development Corporation iii

4 Acknowledgments The successful completion of the project was greatly assisted by discussions with Associate Professor V Ravindran (Massey University, NZ), Dr PH Selle (BASF, Australia), Dr R MacAlpine, Mr G Clatworthy and Ms K Kurko (Ingham Enterprises) and Mr P Pittolo (Weston Animal Nutrition). Dr LI Hew, Ms RJ Gill, Ms M Hayter and Mr K Huang assisted with the conduct of experiments. iv

5 Contents Foreword... iii Acknowledgments... iv Executive Summary... vi 1. Introduction Amino acid digestibility values: a brief review Digestibility assays Variation in digestibility values Application of digestibility values Objectives of studies Animal experimentation Experimental Studies Methodology Bird husbandry Amino acid digestibility bioassays Digestibility calculations Statistical analyses Experiments and results Study 1 - Factors affecting amino acid digestibility Study 2 - Enabling techniques for digestibility bioassays Study 3 - Production cycle performance of broilers fed diets formulated using digestible amino acid values Study 4 - Feed ingredients; amino acid digestibility values Discussion Implications References Appendices Appendix Appendix v

6 Executive Summary The supply of protein and amino acids in poultry diets represents a significant cost of production. The overall objective of this project was to improve the utilisation of dietary amino acids by poultry. One approach to achieving this is to know the quality of available amino acids in the feed. Assessing ileal amino acid digestibility is considered a reasonable method of estimating availability. Digestible amino acid values are becoming important as the basis of poultry feed formulations. However, few commercial nutritionists formulate diets based solely on digestible amino acids. The major reasons include: (a) wide variations in published digestible amino acid values from different sources, arising from differences in sample variation, type of birds, assay diets and assay methodology; (b) insufficient knowledge of the batch-to-batch variation of amino acid digestibility values; and (c) limited published information on broiler responses to diets formulated on the basis of digestible amino acids. Using a series of growth assays, this project addressed these limitations and demonstrated that differences in digestible amino acid content result in comparable differences in broiler performance. Specifically, broilers fed diets formulated on a digestible amino acid basis had significantly improved performance and breast meat yield. Meat yield was further increased by additional dietary methionine supplementation. Age of bird, feed enzymes and feed ingredient processing were all shown to influence the ileal amino acid digestibility of feedstuffs. The outcomes of the project indicate that formulation of broiler diets on a digestible amino acid basis will improve bird performance and increase carcass yield, permit higher dietary inclusion of cheaper, alternative protein sources and decrease nitrogen excretion by the bird. Optimum bird performance and profitability depend largely on adequate and consistent amino acid content of diets. To achieve this goal, ongoing research is required on amino acid digestion and utilisation. Outcomes of such research will reduce nitrogen pollution from poultry production units and increase industry profitability through savings in feed costs. vi

7 1. Introduction 1.1 Amino acid digestibility values: a brief review An important feature of protein quality for the feed industry is knowledge of the availability of amino acids in feedstuffs. Reliable values will permit more efficient formulation of diets. Many attempts have been made to determine amino acid availability (defined as that proportion of dietary amino acids that is in a form suitable for digestion, absorption and utilisation) using in vitro (enzymatic and chemical assays), indirect (microbiological or plasma amino acids) or direct (growth and digestibility assays) methods (reviewed by Ravindran and Bryden, 1999a). Studies in this report were confined to digestibility assays Digestibility assays The digestibility assay has become the most favoured technique for estimating availability, largely because the values apply directly to the bird and all amino acids can be measured in the one assay. Digestibility assays are applied assuming that the difference between input and output is a valid indicator of bioavailability and that digestibility is likely to be the rate limiting step in amino acid availability. Digestibility assays may be divided into faecal and ileal procedures. Excreta digestibility Excreta digestibility has been used by many workers because of its simplicity. Estimates of amino acid absorption made by using excreta of intact birds are in error because avian urine contains some amino acids (Sibbald, 1987). However, the very low concentrations of amino acids in urine mean that the error is likely to be small. Determination of amino acid digestibility in excreta has been widely criticised because intestinal microflora in the hindgut have a substantial affect on the amount of individual amino acids excreted in faeces. Some estimates put this as high as 25% of excreta protein (Parsons et al., 1982). Caecetomised birds were used to overcome the problem of microbial modification of dietary protein and microbial protein synthesis in the hindgut. However, the influence of caecetomy on apparent amino acid digestibility appears from the literature to be quite variable (Ravindran and Bryden, 1999a). Nevertheless, the excreta method using precision-fed roosters has been widely adopted in Canada, the United States and France and in the latter two countries the birds are caecetomised. In this procedure true amino acid digestibility is determined after correction for endogenous amino acid secretion into the gut. Ileal digestibility Since microbial activity is concentrated in the hindgut and the main sites of absorption of amino acids are the jejunum and ileum, Payne et al. (1968) suggested that the analysis of ileal contents rather than excreta might be a reliable method for assessing protein and amino acid digestibility. Ileal digestibility can be determined in two ways depending on the technique of sample collection. The simplest method for the collection of ileal digesta is to kill the bird and the alternative is to use an ileal cannula. Ileal cannulation has been developed for adult cockerels (Rajaho and Farrell, 1984; Gurnsey and James, 1985). Although ileal cannulation seems to provide some theoretical advantages over the other method it is a sophisticated technique for practical application. Some questions may arise such as the rejection of the cannula, the type and placement of the cannula, the free flow of digesta through the cannula or the use of an appropriate marker (see Sauer et al., 1989). Moreover, for the cannulation technique to be cost effective, it must be undertaken with adult birds and there always therefore remains a question as to whether digestibility measured with adults may not reflect digestibility in the rapidly growing broiler chicken. It is for these reasons that, at the University of Sydney, an ileal digestibility assay with five 1

8 week old broiler chickens has been developed and a monograph published that contains the digestibility of 92 samples representing 23 feedstuffs (see Ravindran et al., 1998a) Variation in digestibility values A number of factors influence amino acid digestibility. The nature and digestion of dietary protein will reflect breeding programs, agronomic conditions, presence of anti-nutritive factors and processing. Variation in digestibility values will also arise from difficulties associated with the conduct of assay procedures and the measurement of endogenous amino acid losses. Surprisingly, there are few instances in the literature where the significance of these sources of variation has been evaluated. Dietary protein digestion All dietary sources of protein are heterogenous mixtures of different proteins. It would be anticipated, therefore, that different proteins would be digested at different rates and this in turn would cause a variation in the rate at which different amino acids were taken up from the gut. However, the situation is more complex than this as proteins, although different in their chemical compositions, are not isolated entities but have various linkages with carbohydrates, lipids and other proteins so that these interactions and the composition of the diet may affect the digestibility of dietary protein (Hughes and Choct, 1999). In addition, digestion and absorption may be inhibited by the presence of anti-nutritive factors in the diet. Protease inhibitors, lectins, polyphenolic compounds, saponins and non-starch polysaccharides are examples of anti-nutritive factors that depress protein digestion and utilisation (Bryden, 1996; Hughes and Choct, 1999). Ironically, those feedstuffs (such as grain legumes and oil seed meals) which are used extensively as sources of dietary protein also contain the highest concentrations of anti-nutritional factors. For example, soyabean meal contains a range of anti-nutritional factors, many of which are heat labile and destroyed during feedstuff manufacture (Dale, 1996). Heat treatment, essential for inactivation of many anti-nutrients, may reduce protein quality in the presence of carbohydrates by Maillard type reactions. Processing, especially heat treatment, may contribute to the variability of ingredients such as protein meals and cottonseed meal (Dale, 1996). Lysine is heat sensitive and the low digestibility of lysine in cottonseed meal may reflect heat processing of the meal. The variations in digestibilities of amino acids in meat meals are likely to be due to differences in raw ingredients, time between slaughter and rendering and the duration and temperature of the rendering process (Skurray, 1974). Obviously, optimum processing conditions for all protein meals that do not reduce amino acid digestibilities need to be established. Another aspect of processing, grinding, modifies particle size and shape without causing chemical changes in feedstuffs. It has been shown that grinding improves nutrient digestibility in birds (Hamilton, 1995). This may reflect the increased surface area accessible to enzyme attack during digestion. It has been known for some time that the major influence of anti-nutritional factors on protein nutrition has been a reduction in apparent protein digestibility. It is only recently that the actual cause of the reduction in apparent digestibility has been determined with any certainty. The application of new techniques for the measurement of endogenous amino acid excretion has allowed researchers to separate the effects of reduced digestion of both exogenous and endogenous protein and increased endogenous secretion (Angkanaporn et al., 1994). Both factors would reduce apparent digestibility. The relative importance of these two avenues of amino acid loss by the bird will vary with different anti-nutritive factors (Bryden, 1996). The application of feed enzymes to poultry diets has also demonstrated the impact of anti-nutritive factors on apparent amino acid digestibility. In a series of studies, Ravindran and Bryden (l999b) have 2

9 shown that the application of xylanase and phytase alone and in combination improves amino acid digestibility by amounts that can be quite significant in terms of overall feed formulation. The positive effect of enzymes on amino acid digestibility again demonstrates the impact of anti-nutritive factors on either reducing protein digestion or increasing endogenous amino acid loss. The net result is a decrease in apparent amino acid digestibility. Assay procedures There are now a number of reference sources of known digestibility values for a range of feedstuffs. However, when these compilations are examined it is difficult to know how to compare the values obtained by different procedures. Values have been derived using a number of different assay procedures that vary in terms of the age of the birds used, the collection site of digesta, feeding procedures, basal diet, dietary inclusion level of test ingredients, etc. (see Ravindran and Bryden, 1999a) which all add to the uncertainty of the values obtained. Difficulties associated with amino acid analysis can be a major source of variation which is often overlooked (Ravindran and Bryden, 1999a). Moreover, the application of rapid techniques such as NIR is dependent on the reliability of chemical analysis of amino acids. Two major areas of contention in digestibility assays are the use of ileal versus excreta collection procedures and the correction of digestibility values for endogenous secretions. There have been few direct comparisons of ileal versus excreta digestibility methods, but in a series of studies (Ravindran et al., 1999) it was shown that there is greater variation in excreta values than there is in ileal values. Differences observed between ileal and excreta digestibilities in these studies clearly demonstrated that amino acid metabolism by hindgut microflora in chickens may be substantial and that digestibilities determined at the terminal ileum are more accurate estimates of amino acid availability than those determined in excreta. If feed intake is low, as it is in precision-fed assays, endogenous amino acids become a greater proportion of the amino acids measured in digesta and excreta. Apparent digestibility is depressed accordingly. The problems associated with the quantification of endogenous amine acids are discussed below. Endogenous amino acid losses Most excreta digestibility assays and some ileal digestibility procedures include a correction for endogenous amino acids in an endeavour to provide a more accurate value for comparing different diets or protein sources. Approaches to the estimation of endogenous amino acids in poultry (see Angkanaporn et al., 1996a) have included the measurement of amino acids in excreta either during starvation, when fed a protein free diet, or by determining endogenous output at zero intake by regression analysis. However, the use of these practices, especially the first two, is intrinsically unsound because starvation or the absence of a nutrient, such as protein, profoundly alters metabolism and the bird can no longer be regarded as physiologically normal. Starvation or feeding a protein free diet are the methods used for endogenous correction in the precision-fed rooster excreta digestibility assay which has been adopted in many laboratories. At the University of Sydney, both the protein free diet and the regression analysis method have been used to measure the entry of endogenous amino acids into the lower ileum of broilers and roosters, and these studies have shown that the two methods give different results that vary with the maturity of the bird. These techniques have been compared to the homoarginine method and it has been shown that both techniques significantly underestimate endogenous amino acid secretion when compared with the latter technique (Siriwan et al., 1994). Bryden et al. (1996) and Ravindran and Bryden (1999a) have discussed in detail the assumptions that are used when applying the homoarginine technique and these assumptions have been shown to be valid when tested. Interestingly, the values obtained by the homoarginine technique have been reported to be of similar magnitude to those measured using isotope dilution (Roos et al., 1994). These techniques have 3

10 the advantage that they measure endogenous amino acids in birds that can be considered physiologically normal Application of digestibility values The major advantage of using digestible amino acids in diet formulation is that it makes it possible to increase the inclusion levels of alternate ingredients (in particular, low quality protein sources) in poultry diets. In effect, it will increase the range of ingredients that can be incorporated, improve the precision of formulation and ensure more predictable bird performance. In a series of studies evaluating canola meal (Ravindran et al., 1998b), cottonseed meal (Ravindran and Bryden, l999c) and meat and bone meal (Ravindran and Bryden, 1999d), the beneficial effects of using apparent ileal digestible amino acids in broiler diet formulations to increase the inclusion levels of poorly digestible ingredients were demonstrated. In these studies, as expected, increasing dietary levels of canola meal, cottonseed meal and meat and bone meal on a total amino acid basis significantly lowered weight gains and feed efficiency of broilers. The observed depressions were, however, largely overcome when the diets were balanced on a digestible amino acid basis. This is in accord with previous studies on cottonseed meal (Fernandez et al., 1995) and several byproduct ingredients (Rostagno et al., 1995; Douglas and Parsons, 1999). These results confirm that the inclusion levels of poor quality protein sources in broiler diets can be increased as long as they are based on amino acid digestibility values. Additivity of digestible amino acids, determined in single feedstuffs, is a crucial consideration in the formulation of complete diets. Studies by Angkanaporn et al. (1996c), found that digestible amino acid supply in a complete diet can be predicted, with reasonable accuracy, based on apparent amino acid digestibilities determined for individual feed ingredients (soybean meal, sunflower meal, meat and bone meal). Investigations with a wider variety of ingredients may be warranted to determine the possibility of associative effects between other feedstuffs. A question often posed by commercial nutritionists concerns which digestible amino acid system is most appropriate for use in the formulation of poultry diets - apparent or true digestibility values. Apparent digestibility measures the digestibility of amino acids of both dietary and endogenous origins. True digestibility, on the other hand, includes a correction for endogenous amino acid secretions. The relative merits of these two systems have been discussed in detail by Ravindran and Bryden (1999a). It would appear that the choice of the appropriate system of digestible amino acids may depend on the method of formulating diets. If diets are being formulated on a least-cost basis using linear programming, then apparent ileal digestibility values are the most appropriate as they take into account the endogenous cost of digestion. On the other hand, if diets are being formulated in computer simulation models, then true digestibility values will be relevant as the model should correct for the endogenous cost of digestion. It should be appreciated, however, that both digestible amino systems are superior to the total amino acid system currently employed in practical feed formulations and that all current methods of amino acid evaluation have specific applications and shortcomings. 1.2 Objectives of studies The objective of the studies undertaken within this project was to investigate means of improving the efficiency of amino acid utilisation in meat chickens by (i) adjustment of amino acid intake to meet the birds requirements by formulating diets on a digestible amino acid basis, and (ii) identifying factors that contribute to observed variability in amino acid digestibility. 4

11 1.3 Animal experimentation Experimental procedures described in this report which involved the use of birds were approved by the University of Sydney Animal Care and Ethics Committee and complied with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. 5

12 2. Experimental Studies 2.1 Methodology Bird husbandry Day-old, male broiler chicks were obtained from a commercial hatchery. All birds were reared to 21 days of age in tiered battery brooders. A temperature of 32 ± 1 C was maintained during the first week and gradually decreased to approximately 23 C by the end of the third week. On day 21, they were transferred to Harrison carry on cages housed in an environmentally controlled room (23 C). Chickens were fed a commercial starter diet to 21 days of age and then a commercial finisher diet unless on experiment. Diets were offered ad libitum and water was available at all times Amino acid digestibility bioassays Different assay diets were used for cereal grains and protein meals (Table 1). In the case of cereals, assay diets contained, per kg: 918 g of test cereal, 20 g of vegetable oil and 42 g of mineral and vitamin supplements. For protein meals, assay diets were based on dextrose and contained the test feedstuff as the only source of protein. The proportions of dextrose and the test feedstuff were varied in each diet to obtain 200 g crude protein/kg. Diets containing ingredients of plant origin, blood meal and feather meal had identical calcium and phophorus supplementation, but these supplements were not included in diets containing fish meal, meat meal or meat and bone meal. Solkafloc (30 g/kg) was added as a source of fibre in diets containing animal protein meals. Celite (20 g/kg) was added to all diets as a source of acidinsoluble ash (AIA) which was used as an indigestible marker in the calculation of digestibility coefficients. Each assay diet was offered ad libitum to three pens (4 birds/pen) of male broilers from 35 to 42 days of age unless otherwise stated. At the end of the bioassay, all birds were euthanatised by an intracardial injection of sodium pentabarbitone solution, and the contents of the lower half of the ileum were collected by gently flushing with distilled water into plastic containers. The ileum was defined as that portion of the small intestine extending from the vitelline deverticulum (formerly Meckel s diverticulum) to a point 40 mm proximal to the ileo-caecal junction. Ileal digesta of birds within a pen were pooled, frozen immediately after collection and subsequently freeze-dried. Dried ileal digesta samples were ground to pass through a 0.5 nun sieve and stored in airtight containers at - 20 C for chemical analyses. Amino acid concentrations in the diet and ileal digesta samples were determined using cation-exchange column chromatographic procedures with post-column derivatisation and fluorimetric detection of amino acids using 0-phthaldialdehyde as described by Siriwan et al. (1993). Nitrogen (N) content was determined by the method of Sweeney (1989) using a nitrogen determinator (LECO Corporation, St. Joseph, Michigan, USA). Crude protein content of the ingredients were calculated as N x The AIA contents of the diet and ileal digesta samples were measured after ashing the samples and treating the ash with boiling 4 M hydrochloric acid (Siriwan et al., 1993). 6

13 Table 1. Composition (g/kg air dry basis) of diets used in amino acid digestibility assays selected examples. Ingredient Cereals Plant protein sources Animal protein sources Wheat Soyabean meal (48%) Meat meal Feather meal Dextrose Soyabean oil Solkafloc a Celite Dicalcium phosphate Limestone Choline chloride Salt Vitamin and mineral Premix b Total a James River Co., New Jersey, USA. b Each kg of premix contained the following : vitamin A, 2,200 IU.; vitamin D 3, 700 IU.; vitamin E, 4 g ; vitamin K 3, 0.4 g; riboflavin (vitamin B 2 ) 1.6 g ; pyridoxine HCl (vitamin B 6 ) 1 g ; cyanocobalamin (vitamin B 12 ), 3 g ; biotin, 0.02 g ; niacin, 6 g ; thiamine (vitamin B 1 ), 0.3 g ; calcium pantothenate, 3 g ; folic acid, 0.4 g ; antioxidant, 25 g ; manganese (MnO), 15 g ; zinc (ZnO), 10 g ; iron (FeSO 4.H 2 O), 4 g ; copper (CuSO 4.H 2 O), 1 g ; iodine (Ca(IO 3 ) 2 ) 0.2 g ; cobalt (CoCO 3 ), 0.06 g ; selenium (Na 2 SeO 3 ), 0.02 g ; molybdenum (Na 2 MoO 4 ), 0.32 g. Choline chloride and salt were obtained locally Digestibility calculations The apparent ileal amino acid digestibilities were calculated as follows using AIA as a marker. Amino acid digestibility coefficient = (AA / AIA) d - (AA / AIA) i (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. Digestible amino acid concentrations were calculated from total concentrations and respective digestibility coefficients. Digestible amino acid content = Total amino acid content x Digestibility coefficient 7

14 2.1.4 Statistical analyses The statistical methods used were paired t-test, analysis of variance (ANOVA) and regression analysis by the Minitab program version 11.0 (Minitab, 1996). For ANOVA, multiple comparison of means was performed by Least Significant Difference (LSD) test with a significant level at P < Experiments and results Study 1 - Factors affecting amino acid digestibility As described in Chapter 1, a number of factors have been identified which may alter amino acid digestibility. In this study, a number of experiments were conducted that examined some of these factors. Broiler age and amino acid digestibility The influence of age on protein digestion in broiler chickens is not clearly established. It maybe an important factor in the further refinement of diet formulation using amino acid digestibility values. In the present experiment, the apparent ileal digestibility of amino acids in eight feed ingredients was determined at three ages (14, 28 and 42 days post-hatching). The ingredients were bioassayed using the procedures described above. Assay diets contained the test ingredient as the only source of protein. Following overnight fasting, each assay diet was fed ad libitum to five pens (12 birds/pen at 14 days, 8 birds/pen at 28 days and 6 birds/pen at 42 days) of male broilers for three days. At 14, 28 and 42 days, digesta contents from the terminal ileum were collected and analysed. The average amino acid digestibility coefficients for the three age groups are shown in Table 2. In the Table the reference value is taken from Ravindran et al (1998) and is included for comparison. Values for individual amino acids in each ingredient appear in Appendix 1 (Tables A1 to A8). Table 2. Effect of the age of broiler chickens on the average apparent ileal digestibility coefficients of different feed ingredients in Experiment 1. Maize Sorghum Wheat Millmix Soyabean meal Canola meal Cottonseed meal Meat & bone meal Reference 1 14d 28d 42d SEM b 0.79 ab 0.78 a 0.61 b 0.85 b 0.80 b b 0.83 a 0.77 b 0.72 b 0.62 b 0.87 a 0.81 a a abc Means in a row bearing different superscripts are significantly different (p< 0.5) 0.83 a 0.81 a 0.72 b 0.74 a 0.87 a 0.81 a ab 1 Ravindran, V., Hew, L.I. and Bryden, W.L. (1998). Digestible Amino Acids in Poultry Feedstuffs. RIRDC, Canberra and the Poultry Research Foundation, The University of Sydney, Camden. The influence of age on ileal digestibility varied with different ingredients, but for most ingredients digestibility improved with the age of broiler chickens. The exceptions were wheat where digestibility decreased with age and cottonseed meal where digestibility remained low throughout the production cycle of the broiler. 8

15 Feed enzymes and amino acid digestibility The second factor examined in this study was feed enzymes. Broiler diets, especially wheat-based diets, usually contain an exogenous xylanase and there is increasing evidence that an exogenous microbial phytase may also improve the digestibility of energy and amino acids of these diets. The object of experiment 2 was to determine the influence of a commercial xylanase (Natugrain Blend ; BASF, Ludwigshafen, Germany) 1, and a microbial phytase (Natuphos ; BASF) 2 alone and in combination, on the apparent amino acid digestibility of wheat, sorghum, soyabean meal, canola meal, cottonseed meal, and lupins (L. angustifolius). The amino acid digestibility bioassay was conducted as described in section 2.1. The 25 experimental diets were fed in a mash form to three replicate pens (6 male birds per pen) for five days. On day 42, ileal digesta was collected for digestibility determination and the average values appear in Table 3. Values for individual amino acids in each ingredient appear in Appendix 2 (Tables A9 to A14). Table 3. Effects of xylanase and phytase on the average apparent ileal digestibility coefficients of amino acids in different feed ingredients in Experiment 2. Ingredient Control Xylanase Phytase Xylanase + Phytase Sorghum Wheat 0.72 c 0.79 a 0.75 b c 0.77 a b Soyabean meal Canola meal 0.72 b 0.74 b 0.76 a 0.74 a b Cottonseed meal Lupins 0.82 a 0.77 b 0.82 a b 0.80 a b abc Means within rows with different superscripts differ significantly (P < 0.05) SEM In this experiment enzyme supplementation was without effect on sorghum, soybean meal and cottonseed meal. Xylanase had a positive effect (P < 0.05) on wheat digestibility but a negative effect (P < 0.05) with lupins. Phytase significantly (P < 0.05) improved canola meal digestibility. Ingredient processing and amino acid digestibility Processing of feed ingredients prior to incorporation into the diet is another important factor that may modify amino acid digestibility. The acreage of canola grown in Australia is increasing. The development of low erucic acid, low glucosinolate canola cultivars has led to the availability of an animal feed ingredient of high value after the extraction of oil. The major methods of extracting canola oil are either through expeller or solvent extraction. The aim of the third and fourth experiments was to compare canola meal prepared by both extraction procedures. In Experiment 3 the apparent ileal digestibility of amino acids in both meals (Table 4) was determined as described above using broilers that were five weeks of age. The results of Experiment 3 show that apparent ileal amino acid digestibility was higher in canola, solvent extracted, than in canola, expeller extracted. 1 Natugrain Blend contains xylanase (55,000 EXU/g) and 9 β-glucanase (1,200 BGU/g) and several side- 9 activities (cellulase and protease); the inclusion rate recommended is also 120 g/tonne. 2 Natuphos contains 5,000 FTU/g phytase and for broilers the inclusion level recommended by the manufacturer is 120 g/tonne. One unit of phytase (FTU) is defined as the quantity of enzyme that releases 1 µmol of inorganic phosphorus/min from mol/l sodium phytate at ph 5.5 at 37 C.

16 Table 4. Apparent ileal amino acid digestibility (%) of canola meal expeller and canola meal solvent (Experiment 3). Canola expeller Canola solvent SEM P value Arginine 77.4 b 83.9 a Histidine 73.8 b 79.0 a Isoleucine 70.6 b 75.6 a Leucine 74.5 b 79.4 a Lysine 71.0 b 75.4 a Methionine Phenylalanine 73.3 b 78.6 a Threonine 63.5 b 69.1 a Valine 69.2 b 74.2 a Alanine 74.2 b 78.4 a Aspartic acid 66.8 b 72.5 a Glutamic acid 80.1 b 85.4 a Glycine 70.5 b 73.9 a Serine 64.8 b 71.7 a Tyrosine 69.7 b 76.6 a Total Aamino acids 72.0 b 77.5 a Nitrogen 67.8 b 73.2 a a,b Values in the same row with different superscripts differ significantly (P<0.05). The amino acid digestibility values determined in the above experiment were then used in Experiment 4 to compare both meals in diets that were formulated using total and digestible amino acid values. It is obvious from Table 4 that there are significant differences between canola, solvent extracted, and canola expeller extracted, in terms of apparent ileal amino acid digestibility for broilers. The objective of Experiment 4 was to evaluate the performance from 3 to 40 days of broiler chickens fed diets containing graded levels of canola meal, solvent or expeller, formulated using total (TAA) or digestible amino acid (DAA) values. In formulating the diets, apparent ileal digestible lysine requirement was assumed to be 88% of the total requirement, and the ideal protein concept was applied to estimate the requirements for the other essential amino acids. The inclusion levels of each meal were 0, 150, 300 and 450 g/kg of diet. All ingredients used in this study had previously been assayed for total and apparent ileal digestible amino acids as described above. The results of the growth study are shown in Table 5. In general, birds fed solvent extracted canola meal performed better than birds fed expeller extracted canola meal. Canola, solvent extracted, inclusion up to 300g/kg diet did not significantly impair body weight gain, feed intake or feed efficiency of birds fed diets formulated using digestible amino acid values. In contrast bird performance on expeller meal diets was reduced at inclusion levels greater than 150 g/kg. These differences were reflected in a significant interaction between level of inclusion and processing method. The reasons for the different bird responses to solvent and expeller meals remains to be determined. With both meals, inclusion in diets on an amino acid ileal digestibility basis gave superior (P<0.001) bird performance than inclusion on a total amino acid basis. 10

17 Table 5. Growth, feed intake and feed efficiency of broiler chicks fed diets containing graded levels of canola meals (expeller or solvent extracted) formulated using total and digestible amino acid values (Experiment 4). Level Formulation Growth Intake g feed Treatment (g/kg) (g/b) (g/b/d) g gain Control 0 TAA DAA Expeller 150 TAA DAA Expeller 300 TAA DAA Expeller 450 TAA DAA Solvent 150 TAA DAA Solvent 300 TAA DAA Solvent 450 TAA DAA SEM LSD P value < < < Main effects Processing Expeller Solvent SEM LSD P value < < < Levels SEM LSD P value < < < Formulation TAA DAA SEM LSD P value < Interaction Processing x levels P value Processing x formulation P value Levels x formulation P value Processing x levels x formulation P value

18 2.2.2 Study 2 - Enabling techniques for digestibility bioassays Digestibility bioassays are time consuming and expensive. Given the significance of amino acid digestibility values for efficient feed formulation, it is important that the assay technique be evaluated as new technologies become available. For this study, two alternate approaches to different aspects of the bioassay were examined. Comparison of digestibility markers Acid insoluble ash (AIA) is used routinely as an indigestible marker in poultry digestibility studies. Accurate gravimetric determination of AIA requires a large sample size (2g for diet, 1.2g for digesta). Shortage of the amount of digesta collected in some experiments can limit the number of nutrients analysed. Alkanes, which are long-chain hydrocarbons (C 36 ), have been successfully used to estimate digestibility and plant species selection by herbivores. Only small amounts of samples ( g) are required for alkane analysis and quantification is by capillary gas chromatography. The objective of Experiment 5 was to compare C 36 and AIA as markers to estimate nitrogen and amino acid digestibility in different portions of the gastrointestinal tract of meat chickens. Grower diets were used and contained either celite (as a source of AIA, 20 g/kg), which replaced part of the wheat and sorghum components of the diet (Diet 1), or C 36 (200 mg/kg; Diet 2). The diets were fed ad libitum to six pens of broilers (7 birds/pen) from 25 days of age. After ten days of feeding, digesta from the jejunum and upper and lower ileum was collected following lethal injection of the birds with sodium pentobarbitone. Diets and digesta samples were analysed for nitrogen, amino acids, AIA and C 36, and digestibility calculated. The results for nitrogen and amino acid digestibility (Mean ± SD) are summarised in the Tables 6-9. Table 6. Nitrogen digestibility coefficients in different segments of the gastrointestinal tract (Experiment 5) AIA C 36 Pooled SEM P value Jejunum 0.59± ± Upper ileum 0.72± ± Lower ileum 0.74± ±

19 Table 7. Amino acid digestibility coefficients in the jejunum (Experiment 5) Diet 1 (AIA) Diet 2 (C 36 ) Pooled SEM P value Asp 0.58 ± ± Thr 0.55 ± ± Ser 0.60 ± ± Glu 0.70 ± ± Gly 0.57 ± ± Ala 0.57 ± ± Val 0.60 ± ± Met 0.83 ± ± Ile 0.60 ± ± Leu 0.59 ± ± Tyr 0.61 ± ± Phe 0.63 ± ± His 0.65 ± ± Lys 0.70 ± ± Arg 0.70 ± ± There were no significant differences in the digestibilities of nitrogen or all amino acids when using AIA or C 36 as markers in the three segments of the gastrointestinal tract examined. It is interesting to note that the variations within treatment tend to be smaller for C 36 than AIA as indicated by smaller standard deviations in C 36 than AIA. The results of this study demonstrated that C 36 is a suitable marker to estimate ileal digestibility in poultry. In conjunction with this study, total excreta voided was collected. Digestibility determined with total collection and AIA gave similar values which were significantly different from values determined with C 36. We do not have an explanation for this discrepancy. Table 8. Amino acid digestibility coefficients in the upper ileum (Experiment 5) Diet 1 (AIA) Diet 2 (C 36 ) Pooled SEM P value Asp 0.71 ± ± Thr 0.69 ± ± Ser 0.74 ± ± Glu 0.80 ± ± Gly 0.72 ± ± Ala 0.72 ± ± Val 0.74 ± ± Met 0.91 ± ± Ile 0.74 ± ± Leu 0.75 ± ± Tyr 0.75 ± ± Phe 0.77 ± ± His 0.77 ± ± Lys 0.80 ± ± Arg 0.82 ± ±

20 Table 9. Amino acid digestibility coefficients in the lower ileum (Experiment 5) Diet 1 (AIA) Diet 2 (C 36 ) Pooled SEM P value Asp 0.72 ± ± Thr 0.69 ± ± Ser 0.75 ± ± Glu 0.82 ± ± Gly 0.73 ± ± Ala 0.73 ± ± Val 0.74 ± ± Met 0.92 ± ± Ile 0.73 ± ± Leu 0.76 ± ± Tyr 0.76 ± ± Phe 0.78 ± ± His 0.78 ± ± Lys 0.80 ± ± Arg 0.93 ± ± Comparison of endogenous amino acid markers Endogenous amino acid (EAA) losses in poultry have traditionally been determined by the measurement of amino acid excretion in fasted birds or in birds fed protein-free diets However, these techniques have been criticised because starvation or the absence of protein will cause a reduction of digestive secretions, resulting in the underestimation of EAA losses Two new methods, namely guanidination and hydrolysed casein, which overcome this limitation, are now available. The aim of Experiment 6 was to compare the protein-free diet (PFD), guanidinated casein (GC) and enzymically hydrolysed casein (EHC) methods for determining EAA losses in broilers. The PFD was based on dextrose. The other two diets were based on dextrose and GC or EHC to give a protein level of 200 g/kg, and also contained 20 g/kg celite as a digesta marker. Each test diet was fed ad libitum to three pens (6 birds/pen) of male broilers from 35 to 38 days of age. On day 38, lower ileal contents were collected. The diets and digesta were analysed for amino acids (including homoarginine in the GC treatment) and acid-insoluble ash, and the EAA losses were calculated as previously described (Butts et al., 1992; Siriwan et al., 1994) and are presented in Table 10. The EAA losses determined with the use of PFD were considerably lower (P < 0.05) than those determined by GC and EHC methods. The total losses of EAA from GC and EHC methods were almost three-fold greater (P < 0.05) than those determined by the PFD. The EAA flow values obtained from GC and EHC methods were similar (P > 0.05). These results show that EAA corrections based on the PFD method significantly underestimate true digestibility values. 14

21 Table 10. The endogenous flow (g/kg dry matter intake) of selected amino acids determined using a pretein-free diet (PFD), guanidinated casein (GC) or enzymatically hydrolysed casein (EHC) (Experiment 6). Amino acid PFD GC EHC Pooled SEM Aspartic acid 0.61 a 1.59 b 1.43 b 0.12 Threonine 0.51 a 1.44 b 1.09 b 0.14 Glutamic acid 0.72 a 3.56 b 3.87 b 0.31 Glycine 0.36 a 0.69 b 0.49 ab 0.07 Valine 0.42 a 1.06 b 0.94 b 0.08 Isoleucine 0.29 a 0.97 b 0.88 b 0.07 Leucine 0.44 a 1.02 b 1.00 b 0.06 Lysine 0.21 a 0.78 b 1.02 b 0.10 Arginine 0.28 a 0.68 b 0.54 b 0.05 Total 5.26 a b b 0.63 a,b Means in a row bearing different superscripts are significantly different (P<0.05) Study 3 - Production cycle performance of broilers fed diets formulated using digestible amino acid values. It is now generally agreed that bird performance is superior when fed diets based on digestible amino acid values. This is particularly apparent when feed ingredients of low digestibility are incorporated in diets. Most of the studies that have evaluated the use of digestible amino acids in feed formulation have only monitored bird performance during the starter phase. In this study bird performance was measured throughout the production cycle from hatch to processing. The objective of Experiment 7 was to compare the performance of broiler chickens fed diets that were formulated using total amino acid values and compared to: (a) diets formulated using the RIRDC Booklet 1 values for digestible amino acids; (b) diets formulated using ingredients of known amino acid digestibility; and (c) diets formulated by a large integrated broiler company. Diets were formulated for starter, grower and finisher phases using the following assumptions. Total lysine requirement was estimated to be 1.27%, 1.01% and 0.86% for starter, grower and finisher diets, respectively, based on the University of Illinois digestible amino acid requirement of 1.12%, 0.89% and 0.76% of diet, and an assumed apparent ileal digestible lysine requirement of 88% of the total requirement. The ideal amino acid ratio concept was applied to estimate the requirements for the other essential amino acids. All diets were mixed using the same batch of ingredients for which digestibility values had been determined. Each diet was fed to six pens of six male broilers (Cobb); starter from days 1 to 14, grower from days 14 to 28 and finisher from days 28 to 42. Feed consumption and body weight were recorded weekly. On day 42, breast weight and abdominal fat weight measurements were made on 12 birds from each diet following a lethal injection of sodium pentobarbitone. The results are summarised in Table Ravindran, V., Hew, L.I. and Bryden, W.L. (1998). Digestible Amino Acids in Feedstuffs for Poultry. RIRDC, Canberra and the Poultry Research Foundation, The University of Sydney, Camden. 15

22 Table 11. Bird performance (0-42 days) (Experiment 7) Diet 1 Diet 2 Diet 3 Diet 4 SEM P-value Body Weight (g) 2050 a 2426 b 2437 b 2420 b Feed Intake (g/bird) 3800 a 4323 b 4281 b 4287 b Feed Conversion (g/g) 1.89 a 1.83 b 1.80 b 1.81 b Breast muscle 12.2 a 15.4 b 15.6 b 17.3 c (% bodyweight) Abdominal fat pad (% body weight) 2.4 a 2.1 ab 2.0 b 1.5 c a,b,c Values in the same row with different superscripts differ significantly (P<0.05). Final bodyweight (P<0.001), feed intake (P<0.001) and feed conversion ratio (P<0.005) were significantly improved when diets were formulated on a digestible amino acid basis. These diets also significantly increased breast muscle weight (P<0.001) but decreased abdominal fat pad weight (P<0.001). The change in carcass composition was most apparent with birds fed diet 4. The results of this experiment demonstrate that formulation of diets using digestible amino acid values and fed from hatch to processing can significantly improve bird performance and carcass composition. The opportunity was also taken with this experiment to determine how closely the ileal digestibility values determined with individual feed ingredients or taken from published values predict amino acid digestibility of complete diets. At the completion of the growth study (42 days of age) ileal samples were collected from birds fed diets 2 and 3 and amino acid digestibility was determined. The predicted and determined apparent ileal amino acid digestibilities of the diets are shown in Table 12. Although there were large differences in apparent digestibility among amino acids, the results indicate that values are additive and that amino acid supply in a complete diet can be predicted from amino acid digestibilities of individual ingredients. The results of Experiment 7 demonstrate that formulating diets based on digestible amino acid value was superior to formulation of diets using total amino acids. Highest breast yield and lowest abdominal fat content, however, was associated with birds fed the commercial diet which had higher dietary concentrations of lysine and methionine. The objective of Experiment 8 was therefore to determine the optimum levels of lysine and methionine in a broiler diet for producing maximum breast yield and minimum abdominal fat mass when formulated on a digestible amino acid basis. The experimental diets used in Experiment 8 were based on diet 3 of Experiment 7. The dietary levels of lysine and methionine for the diets used are given in Table 13. There were four levels of lysine and three levels of methionine. Each diet was fed to six pens of six birds; starter from days 1 to 14, grower from days 14 to 28 and finisher from days 28 to 40. On day 40, breast muscle yield and abdominal fat mass were measured. 16