Dietary protein and amino acids Consideration of the undigestible fraction 1

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1 Dietary protein and amino acids Consideration of the undigestible fraction 1 Paul J. Moughan,* 2 V. Ravindran, and J. O. B. Sorbara * Riddet Institute, Massey University, Private Bag , Palmerston North 4442, New Zealand; Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Private Bag , Palmerston North 4442, New Zealand; and DSM Nutritional Products, Animal Nutrition & Health, Av. Eng Billings, 1729, São Paulo, Caixa Postal 3003, Brazil INTRODUCTION The domestic chicken does not digest and absorb all of the protein and amino acids present in its diet, and the undigested component is a substrate for microbial metabolism, which occurs mainly in the hindgut. The undigested protein is defined as that amount of dietary protein that has not disappeared during transit of the material through the digestive tract or part of the digestive tract. Following this definition, based on disappearance, protein and amino acid digestibility are measures that are calculated indirectly by difference. The current recommended measure of amino acid digestibility in poultry is standardized true ileal digestibility (SID %, see below), and the amount of amino acids assumed to have been absorbed is the standardized ileal digestible fraction. The undigestible fraction (%) is therefore 100 SID. It is important to understand what influences the undigested component in the bird and how this dietary fraction can be minimized. Determination of Protein/AA Digestibility in Poultry 2014 Poultry Science Association Inc. Received December 26, Accepted May 10, Presented as part of the Symposium: Possible Substrates for Exogenous Enzymes at the Poultry Science Association s annual meeting in San Diego, California, July 22 to 25, Corresponding author: p.j.moughan@massey.ac.nz ABSTRACT A case is made for the application of true ileal amino acid digestibility and true ileal reactive lysine digestibility (lysine availability) in poultry nutrition. Technical aspects of the true ileal digestibility assays are reviewed, as are factors influencing amino acid digestibility in the broiler. There is considerable variation in amino acid digestibility and lysine availability both within and among diverse feedstuffs. Differences in mean amino acid digestibility among feedstuffs, and the variability in the digestibility of an amino acid within a feedstuff should both be taken into account during least-cost dietary formulation. Key words: enzyme, protease, method 2014 Poultry Science 93 : Over the years, numerous methods have been developed to allow determination of dietary protein (nitrogen) and amino acid digestibility in poultry (Papadopoulos, 1985). Such assays usually involve collecting either excreta from intact or cecectomized birds or ileal digesta. A criticism of methods based on the quantitative collection of excreta is that the gut microbiota, resident in the hindgut, influences the amino acid composition of the excreta (Austic, 1983; van Weerden, 1989; Whitacre and Tanner, 1989; ten Doeschate et al., 1993). Also, excreta contain urinary nitrogen and amino acids, which confound excreta-based nitrogen and amino acid digestibility values. With some feedstuffs and amino acids, there may be a net synthesis of amino acids (e.g., methionine) by the bacteria populating the hindgut, but usually there is a net loss of amino acids as they traverse the hindgut. It does not appear as though amino acids per se are absorbed in the hindgut of the chicken in nutritionally significant amounts (Webb, 1990), but there are some reports of cecal (proximal) amino acid absorption (Moreto and Planas, 1989; Stevens and Hume, 1995). The nonabsorption of at least lysine has been confirmed in our own studies (B. Camden, Massey University, Palmerston North, New Zealand, and P. J. Moughan, unpublished data) wherein homoarginine, an analog of lysine, infused into the hindgut of the chicken was not recovered in the blood. It seems that hindgut amino acid absorption has not been studied extensively in the chicken and this needs to be addressed. The respective net breakdown and net synthesis of amino acids by the hindgut bacteria mean that excreta protein is not representative of undigested dietary protein and that digestibility coefficients in the bird, based on amino acids recovered in the excreta, will be in error. 2400

2 To lessen the potential effects of the hindgut microbiota, some researchers have used cecectomized birds in digestibility assays. However, although hindgut microbial activity is reduced following cecectomy, it is not completely eliminated (Whitacre and Tanner, 1989). Payne et al. (1968) avoided the influence of the hindgut microbes by sampling digesta from the terminal ileum of the bird, upon euthanasia. Following this pioneering work, others have also used the ileal technique, often collecting digesta after killing the bird or under anesthesia, though sometimes from the conscious bird following surgical implantation of a cannula (Soares and Kifer, 1971; Varnish and Carpenter, 1975; Achinewhu and Hewitt, 1979; Bielorai et al., 1982; Raharjo and Farrell, 1984; Summers and Robblee, 1985; Johns et al., 1986; Perez et al., 1993; ten Doeschate et al., 1993; Kadim and Moughan, 1997a; Ravindran et al., 1999). With the ileal technique, there may be a confounding effect on digestibility determination due to the activity of bacteria found mainly in the distal ileum. The magnitude of such a potential effect remains to be investigated. Most contemporary assays for ileal digestibility in poultry rely upon sampling digesta at the end of the small intestine, upon euthanasia, and there has been a considerable amount of work undertaken to assess technical aspects of the ileal digestibility assay, such as test diet composition, feeding method, indigestible dietary marker, time and method of killing, method for sampling digesta, length of ileum sampled, and age of bird used (Summers and Robblee, 1985; Yap, 1991; Kadim and Moughan, 1997a,b; Ravindran and Bryden, 1999; Kadim et al., 2002; Kim et al., 2011, 2012a; Poureslami et al., 2012). A particularly important aspect of assay technique is the sampling of the ileal digesta. The bird should be euthanized in such a manner so as to avoid the shedding of mucosal cells and the inter-mixing of digesta from different intestinal segments, and the contents should be gently flushed from the terminal ileum, rather than being extruded manually (Poureslami, et al., 2012). Euthanasia of birds using the barbiturate, sodium pentobarbitone, is likely to be superior to CO 2 asphyxiation or cervical dislocation (Yap, 1991). Attention to detail is required to achieve reliable results with a satisfactorily low degree of variability. Much is known of the factors influencing the ileal digestibility assay with poultry and there is an urgent need for specification of an internationally recognized standard procedure. Such an agreed methodology would allow better comparisons across studies and diverse feedstuffs. With the ileal digestibility assay, the sampled ileal digesta contain a significant amount of endogenous protein and nondietary bacterial protein. The latter are normally referred to collectively as the endogenous component, and clearly this must be corrected for in determining the digestibility of the dietary amino acids. If such a correction is not made, and the dietary AA intake is corrected for the total ileal amino acid SYMPOSIUM: SUBSTRATES FOR EXOGENOUS ENZYMES 2401 output, the amino acid digestibility value determined is referred to as an apparent value. The use of apparent digestibility coefficients is inappropriate on 2 counts. The apparent values (uncorrected for basal endogenous amino acid losses) can be greatly influenced by assay conditions, especially feeding level and dietary amino acid concentrations, but also the basal amino acid loss (a protein maintenance cost) is already included in the amino acid requirement estimate and therefore should not be reflected in the digestibility coefficient. The maintenance cost should not be paid for twice! Endogenous protein at the terminal ileum is considered to consist of 2 components: a basal endogenous flow, which is related to food DM intake and is common to all diets, and a dietary-variable flow, referred to as the specific endogenous protein loss. The specific loss is affected by dietary factors such as type and concentration of fiber and the amounts and types of various antinutritional factors (ANF; e.g., trypsin inhibitor, tannins, lectins). For protein sources not containing fiber or ANF (e.g., casein, gluten), the ileal endogenous flow approximates the basal flow. Several methods have been developed to measure the basal and specific endogenous protein flows (Moughan et al., 1998). Some of these can be used routinely in feedstuff evaluation [e.g., protein-free diet method, regression method, enzyme hydrolyzed protein/ultrafiltration (EHP) method], whereas others (e.g., isotopic labeling of feedstuff or bird, the homoarginine method, natural proteins devoid of specific amino acids) are better suited to experimental applications. With the protein-free diet and EHP (casein) methods, the ileal basal protein flow is determined directly, and when the total ileal flow is corrected for this basal flow, a true coefficient of digestibility is derived. The term true in this context is consistent with the original use of this term. If an agreed constant basal endogenous protein flow (mg/g food DM intake) is adopted, the digestibility coefficient is referred to as standardized digestibility (Stein et al., 2007) and standardized amino acid digestibility coefficients have been found to be superior to their apparent counterparts in poultry diet formulations (Hoehler et al., 2006). If, however, a method for determining ileal endogenous protein flow is applied that measures both basal and specific endogenous protein losses (e.g., 15 N isotopic labeling of soybean protein), and correction for both components (basal and specific losses) is made, then the digestibility coefficient derived has often been referred to as real digestibility (Boisen and Moughan, 1996). The term standardized has been put forward (Stein et al., 2007) to replace the original term true, whereby correction is made for a basal loss, and then the term true is reserved for what has previously been referred to as real digestibility. True (as used in the original sense) or standardized digestibility is usually the best approach for practical dietary formulation for poultry because the extra amino acid costs associated with the specific losses are not normally included in the

3 2402 Moughan et al. Table 1. True digestibility of amino acids in diets containing heat-treated meat and bone meals determined using intact (I) and cecectomized (C) cockerels 1 Amino acid Diet Lysine I C Methionine I C Threonine I C From Johns et al. (1986). 2 Diet 1 contained 10% meat and bone meal ex the processing factory. Diet 2 contained 10% meat and bone meal from the same batch as used in diet 1, but after 5 h heating in a steam-jacketed cooker. estimate of the amino acid requirement of the bird and these costs should be debited against the feedstuff, as happens when true digestibility is used. Attempts have sometimes been made to use constant values for the basal endogenous losses to correct existing apparent digestibility data sets to a standardized basis. This needs to be considered carefully, as the basal losses are affected by factors such as feeding level, BW, dietary protein content, and potentially by factors such as breed, strain, sex, and environment. Furthermore, if apparent data sets are converted in this manner (retrospectively) to standardized values, then the relevant amino acid requirement value should not be adjusted when evaluating the new data set, as the basal loss is already covered in the requirement value, given how amino acid requirement values for birds have been derived. The standardized digestibility values will rightly reflect the cost to the bird of the feedstuff-variable specific endogenous amino acid losses, and the basal loss will be met by the amino acid intake consistent with the requirement value. True (as used in the original sense) ileal amino acid digestibility is considered the current state-of-the-art approach for determining protein and amino acid digestibility in poultry. True digestibility is a fundamental property of the feedstuff, being independent of food intake or the concentration of the protein/amino acid in the test diet. International agreement on a method to determine basal ileal endogenous amino acid flow in poultry, needed to allow a standardized approach to digestibility determination, would be a major step forward. At present, some workers (e.g., Lemme et al., 2004; Garcia et al., 2007) have calculated standardized ileal amino acid digestibility coefficients based on correction for endogenous losses using the EHP/ultrafiltration method (Moughan et al., 1990), whereas others have used endogenous losses after feeding birds a nitrogen-free diet (e.g., Kim et al., 2011). In choosing a method to be applied to derive the agreed basal flows, consideration should be given to the unphysiological nature of protein-free alimentation (Low, 1980), and the published evidence that dietary protein leads to higher endogenous protein flows (Moughan, 2003). The absolute protein contents of the test diet and diet used to determine the endogenous losses should be considered because dietary protein concentration also appears to influence the endogenous ileal amino acid flow (Hodgkinson et al., 2000; Hodgkinson and Moughan, 2007; Ravindran et al., 2009). Ideally, in a broiler ileal digestibility assay, a paired bird (same age, BW, breed, sex, level of food intake, and so on) would be used to derive, experimentally, the basal endogenous amino acids (Hoehler et al., 2006) to allow correction of the total flow in the paired counterpart. Ileal Versus Excreta Amino Acid Digestibility Although there seems to be a sound theoretical basis for the superiority of the ileal amino acid digestibility assay for application with poultry, questions have been raised as to the practical significance of differences between the ileal and excreta based approaches. This is a valid concern because the chicken, compared with other monogastric animals such as the pig, has a relatively short hindgut and a more rapid rate of food passage through the gut. The potential for considerable differences, however, is highlighted by the data of Johns et al. (1986; Table 1), who used intact and cecectomized cockerels to determine amino acid digestibility in normal and experimentally heat-treated meat and bone meals. This is a useful approach, as the effect of the hindgut microbiota is likely to be greatest for feedstuffs of low digestibility. There were statistically and practically significant differences in true amino acid digestibility between intact and cecectomized birds for the basal meat and bone meal with the difference being magnified after heat treatment of the meal. Cecectomized birds were used in this study, and such differences would be expected to be even higher with the ileal digestibility measure. Ravindran et al. (1999) determined the influence of site of measurement on amino acid digestibility for different types of feedstuffs. Selected data from this study are given in Table 2. Apparent ileal amino acid digestibility values in some feed ingredients were similar to the corresponding excreta values, but significantly lower or higher in some others. As expected, digestibility differences between the sites of measurement were found to be more important with more poorly digested feed ingredients. Overall, the data show that amino acid digestibility determined at the terminal ileum is different from that determined at the excreta level. A similar conclusion was reached by Kadim et al. (2002; see Table 3). It is interesting to note that the directions of the quite large differences found for wheat differed between the Ravindran et al. (1999) and Kadim et al. (2002) studies, but the former study reported apparent digestibility coefficients, whereas the later study gave true digestibility values.

4 SYMPOSIUM: SUBSTRATES FOR EXOGENOUS ENZYMES 2403 Table 2. Differences between apparent excreta and ileal amino acid digestibilities (% units) in selected cereals, plant and animal protein meals for broilers (difference = excreta digestibility ileal digestibility) 1 Item Corn Wheat Soybean meal Cottonseed meal Meat meal Fish meal Feather meal Threonine ** +2.4* * +7.3** +24.5* Valine 5.8** 14.6* 5.8* Methionine * * Isoleucine 8.2* ** 3.4** ** Leucine * * Phenylalanine Histidine Lysine * ** +14.0* * Arginine * ** +15.8* Mean * * 1 From Ravindran et al. (1999). P < 0.10; *P < 0.05; **P < Factors Influencing True Ileal Amino Acid Digestibility 1. Influence of Age of Bird. There is a comprehensive literature on the effect of age on amino acid digestibility in the chicken, but results have been conflicting. Part of the confusion arises from the use of different methodologies with chickens of different ages, and that measures of digestibility have been apparent values based on the collection of excreta. The effect of age of bird on ileal amino acid digestibility has been studied less, but the results are also less conflicting. The work of Noy and Sklan (1999) is of particular interest, in that the uptake of labeled free methionine up to the end of the small intestine was determined (true digestibility). Methionine uptake was low at hatch (55%), increasing to close to 80% by d 4. In a related study (Noy and Sklan, 1995), small intestinal digestion of feed nitrogen (corn/soybean meal diet) increased from 78% at d 4 to 92% at d 21. These studies show that digestion may be impaired in chicks younger than 1 wk of age, which is in accordance with the findings of Batal and Parsons (2002). The digestibility coefficients determined by Noy and Sklan (1999) were true values, but often in practice apparent values have been determined, which begs the question of the effect of age of bird on ileal endogenous amino acid losses. Ravindran and Hendriks (2004) applied the enzyme hydrolyzed casein/ultrafiltration method (also referred to as the peptide alimentation/ ultrafiltration method, Moughan et al., 1990) to broiler chickens of 14 and 42 d of age to determine the effect of age on ileal endogenous nitrogen and amino acid flow. There was a statistically significant effect of age on ileal amino acid losses, with endogenous loss increasing with advancing age for most of the amino acids examined. This needs to be taken into account when comparing the effect of age on ileal amino acid digestibility in chickens. An effect of age on ileal endogenous amino acid flows was also reported by Adedokun et al. (2007) after the administration of a protein-free diet to broiler chickens of 5, 15, and 21 d of age. Ileal endogenous amino acid flow was higher for methionine and threonine at d 5, but there were no differences between d 15 and 21. The study of Garcia et al. (2007) is of particular interest. Here, true ileal amino acid digestibility was determined (after euthanasia) for several food ingredients for broiler chickens of 7 and 21 d of age. The same test diets were given to roosters using a precision-fed ce- Table 3. Magnitude of significant (P < 0.05) excreta-ileal differences 1 for true amino acid digestibility values of feed ingredients for broilers 2 Amino acid Sorghum Wheat Soybean meal Meat-and-bone meal Fish meal Blood meal Threonine Valine Methionine Isoleucine Leucine Phenylalanine Tyrosine Histidine Lysine Arginine Aspartic acid Serine Glutamic acid Proline Alanine [(Excreta digestibility ileal digestibility)/ileal digestibility] From Kadim et al. (2002).

5 2404 Moughan et al. cectomized rooster assay. For most amino acids, there was no difference in true ileal amino acid digestibility between birds of 7 and 21 d of age; however, digestibility was lower at 7 d and sometimes at 21 d, compared with the rooster digestibility values. The latter difference may be due to age effects or to differences in assay methodology. In a similar type of study, Adedokun et al. (2009) determined standardized true ileal amino acid digestibility in broilers (euthanasia) and layers (euthanasia) for 5 plant-based feed ingredients and 1 animal-based feed ingredient. The standardized true ileal amino acid values for laying hens were lower compared with broilers for corn, higher for meat and bone meal, but not different for the remaining 4 feed ingredients. The standardized true ileal amino acid digestibility values in cecectomized roosters (precisionfed) were sometimes higher than in broilers, but once again age and method effects were confounded in the latter comparison. Adedokun et al. (2008) determined standardized true ileal amino acid digestibility for 5 plant-based feed ingredients in 5- and 21-d-old broiler chickens and the same aged turkey poults after euthanasia. For the broilers, true ileal amino acid digestibility increased with age when chicks were fed corn distillers dried grains with solubles and corn, but not soybean meal or canola meal. In the turkey poults, there was no effect of age on true ileal amino acid digestibility in all feed ingredients evaluated except corn. Taken together, these results suggest that when correction is made for endogenous ileal amino acid flow, purported age-related effects on ileal protein digestibility often disappear. Protein digestion is impaired in very young birds (<5 d of age), and may also differ between growing birds (broilers) and mature birds (layers and roosters), though solid evidence for the latter conjecture is lacking. It appears that the practice of using amino acid digestibility values generated with broilers for layers may not be appropriate for all feed ingredients and that values obtained using cecectomized cockerels may overestimate amino acid digestibility in the broiler. For the broiler, true ileal amino acid digestibility appears to be rather constant, for a given diet, for much of the growth trajectory. 2. Other Factors Influencing Amino Acid Digestibility. Other factors such as breed/strain, sex, environmental temperature, and immune status may affect protein digestion in the chicken, but little comprehensive information is available based on true ileal amino acid digestibility measures. Having said this, there is some evidence, although certainly not unequivocal, of effects of sex (reported direction of effect variable), breed or strain of broiler, and environmental temperature (digestibility decreasing with higher temperatures) on amino acid digestibility (Wallis and Balnave, 1984; Zelenka and Liska, 1986; Gruhn and Zander, 1989; Mitchell and Smith, 1990; Firman, 1992; Zuprizal et al., 1992, 1993; ten Doeschate et al., 1993; Koelkebeck et al., 1998; Al-Marzooqi et al., 2011; Kim and Corzo, 2012). These various potentially important effectors need to be studied systematically using recently developed true ileal amino acid digestibility assays, before conclusions can be drawn as to effect, and the direction of the effect. There is scant information on the effect of disease stress or social stress on digestion, though an anorexic state often induced by infection (Baker, 2009) may influence amino acid digestion. True Ileal Amino Acid Digestibility Differences Among Feedstuffs One of the more comprehensive data sets on ileal amino acid digestibility in poultry is that of Ravindran and coworkers (over 200 samples representing 31 feed ingredients, Ravindran et al., 1998, 2002, 2005; Bryden et al., 2009; Nalle, 2009). The ingredients assayed included, inter-alia, cereals (barley, corn, sorghum, triticale, and wheat), cereal by-products (rice polishings and wheat middlings), oilseed meals (canola, cottonseed, soybean and sunflower meals), full-fat canola, corn gluten meal, distillers dried grains with solubles, grain legumes (chickpeas, fava beans, field peas, and lupins) and animal protein sources (blood, feather, fish, meat, and meat and bone meals). The mean ileal digestibility coefficients (albeit apparent values; euthanasia method) of amino acids in wheat and corn were higher than those in sorghum, triticale, and barley. However, variations observed in individual amino acid digestibilities among samples within cereal type were greater than those determined between cereals. Threonine and lysine were the least digestible dietary indispensable amino acids in cereal grains. The most digestible dietary indispensable amino acid was phenylalanine in wheat and leucine in corn and sorghum. In the case of the wheat middlings and rice polishings, threonine was the least digestible indispensable amino acid and arginine was the best digested. In the oilseed meals assayed, amino acid digestibility was highest for soybean and sunflower meals, intermediate for canola meal, and lowest for cottonseed meal. Ileal digestibility coefficients of amino acids in grain legumes and distillers dried grains with solubles were found to be generally similar to those in soybean meal. The digestibility of arginine was the highest and that of threonine the lowest of the indispensable amino acids in oilseed meals and grain legumes, except in cottonseed meal. Lysine was the least digestible amino acid in cottonseed meal. In the animal protein sources assayed, digestibility coefficients of amino acids in blood meal were high, intermediate in fish meal, and low in meat meal, meat, and bone meal and feather meal. Variation in amino acid digestibility coefficients determined for blood meal samples was small. However, wide variations in amino acid digestibilities were observed for other animal protein sources, highlighting significant batch-to-batch differences. In particular, marked variations were determined for meat meal and meat and bone meal samples. Cysteine was the least digested amino acid in animal protein meals, with the exception of blood meal in which isoleucine

6 SYMPOSIUM: SUBSTRATES FOR EXOGENOUS ENZYMES 2405 had the lowest digestibility. Variation in the apparent ileal digestibility of amino acids in 19 meat and bone meal samples for broilers was studied by Ravindran et al. (2002). Considerable variation was observed both in the contents of CP (39 67%) and amino acid digestibility (e.g., lysine, 45 82%; cysteine, 15 56%). Kadim et al. (2002), determining true ileal amino acid digestibility in broiler chickens, also reported a relatively low value for amino acid digestibility in meat and bone meal, but also found very low digestibility for blood meal, highlighting the variability in the digestibility of animal protein meals and the importance of optimal processing. Ravindran and Morel (2006) gave data for highly digested proteins, which extend the Ravindran data set. Casein protein was found to be more digestible (apparent ileal digestibility in broiler) than isolated soy protein, which was more digestible in turn than soybean meal. The overall mean apparent ileal amino acid digestibility coefficients for casein, isolated soy protein, soybean meal, corn gluten meal, and spray dried plasma were 0.95, 0.89, 0.82, 0.85, and 0.86, respectively. Another relevant comprehensive data set is that of Lemme et al. (2004), wherein standardized true ileal CP and amino acid digestibility values were reported for broilers (euthanasia method) for a range of ingredients. Among the cereal grain products, corn and wheat had the highest amino acid digestibilities, (e.g., methionine + cysteine: corn = 90%, wheat = 91%), followed by barley and triticale, having generally lower values (e.g., methionine + cysteine: barley = 89%, triticale = 88%) and with sorghum, rice pollard, and wheat middlings having lower amino acid digestibility values (e.g., methionine + cysteine: sorghum = 84%, wheat middlings = 78%, rice pollard = 68%). For the plant protein sources studied, soybean meal, sunflower meal, corn gluten meal and lupins all supported relatively high amino acid digestibilities (e.g., methionine + cysteine: sunflower meal = 87%, soybean meal = 86%, corn gluten meal = 83% and lupins = 85%), whereas cottonseed meal, peas and beans, and canola meal demonstrated considerably lower digestibilities. For the 3 animal meals studied, fish meal had the highest amino acid digestibility with meat and bone meal and feather meal having quite low digestibilities (e.g., methionine + cysteine: fish meal = 82%, meat and bone meal = 62%, feather meal = 51%). A third published data set of digestibility in chickens is that of Rhône-Poulenc (1993), where true amino acid digestibility values were derived using a cecectomized cockerel assay. The trends in the digestibility values for cereals mirror those discussed by Ravindran and coworkers. Amino acid and protein digestibility in wheat and corn were generally higher than in barley, sorghum, triticale, and rye, though the protein digestibility of sorghum was highly dependent upon the cereal s tannin content (Table 4). The lower digestibility of the hightannin sorghum will be due in part to a lowered digestion of the sorghum protein, but will also be influenced by enhanced endogenous protein losses. This comparison is an excellent example of the effect of ANF (tannins) inducing specific ileal endogenous amino acids, thus leading to lowered true digestibility values. Rhône- Poulenc (1993) report moderate digestibility values for wheat middlings and wheat shorts but much lower values for wheat bran and rice bran. The lower amino acid digestibility values for the brans undoubtedly reflect their higher fiber content. Corn gluten meal, corn gluten feed, and corn germ meal gave relatively high digestibilities whereas amino acid digestibility in corn distillers and solubles and bakery by-products was considerably lower. The amino acid digestibility of soybeans was found to be dependent upon the type of processing (Table 4), a result confirmed more recently by Clarke and Wiseman (2007), who found an effect of extrusion conditions on the apparent ileal digestibility of lysine and other amino acids in full-fat soybeans. Lupins had high digestibilities, with lower values for extruded rapeseed, field peas, and horsebeans and particularly low values (50 70% digestibility) for alfalfa (lucerne) meal. The digestibility of rapeseed meal was dependent on the amount of glucosinolate present (Table 4). Cottonseed meal, linseed meal, and coconut meal all gave low amino acid digestibilities, but the protein in groundnut meal, sesame meal, and sunflower meal was well digested. For the animal-based meals, high amino acid digestibilities were reported for fish meal and fish solubles, intermediate values for bone meal and blood meal, and low values for meat and bone meal, poultry by-product and feather meal. Variation in amino acid digestibility within an ingredient is just as important as variation among different ingredients. Rhône-Poulenc (1993) gives useful data on variation around the determined mean true amino acid digestibility coefficients (Table 5). Variation around the mean true digestibility value was generally lower for CP compared with individual amino acids and tended to be higher for threonine and cysteine in comparison with lysine and methionine. Feedstuffs such as cottonseed meal and some of the animal by-products were particularly variable. Such variation should be taken into account in valuing the price of a food for dietary formulation. Taking the 3 data sets together, a pattern for differences in amino acid digestibility across feedstuffs begins to emerge, giving some credence to the use of average digestibility values, but it must be borne in mind that the variation in amino acid digestibility within a feedstuff may be greater than the variation among feedstuffs. Ideally, a rapid in vitro method for predicting digestible amino acids should be available to reliably provide information that could be used in diet formulations. Unfortunately, and despite much work in this area, no such method has been forthcoming. Such a method is urgently needed, but in the meantime mean digestibility values should be used and the degree of variation in amino acid digestibility should be taken into account in determining the cost of feedstuffs during feed formulation.

7 2406 Moughan et al. Table 4. True amino acid digestibility of sorghum grain, processed soybeans, and rapeseed meal determined with cecectomized cockerels 1 Digestibility (%) Amino acid Sorghum 1 (tannins < 0.5%) Sorghum 2 (tannins > 0.5%) Soybean 1 2 (toasted) Soybean 2 2 (extruded) Rapeseed 1 (glucosinolate < 20 μm/g) Rapeseed 2 (glucosinolate > 20 μg/g) Protein Lysine Methionine Cysteine Threonine Tryptophan Arginine Glycine Serine Histidine Isoleucine Leucine Phenylalanine Tyrosine Valine From Rhône-Poulenc (1993). 2 Full fat soybean. In more recent investigations, Szczurek (2009) undertook a true ileal digestibility study (anesthesia) with 30-d-old broilers, where ileal digestibility coefficients were standardized using age-specific ileal endogenous amino acid losses (protein-free diet). Some of the digestibility values obtained for the birds were quite low. In general, the true ileal amino acid digestibilities of the various feedstuffs examined ranked: corn > wheat > barley (within cereals); fava beans = peas > rapeseed (within native seeds); soybean meal > rapeseed expeller cake > solvent extracted rapeseed meal > corn distillers dried grains with solubles. In a companion study, but with younger birds (14-d-old chicks), Szczurek (2010) found a different ranking for the cereals, with wheat being superior to corn. Soybean meal was found to be generally superior to full-fat rapeseed, rapeseed cake, and solvent-extracted rapeseed meal. Corn distillers dried grains with solubles was the least digestible material evaluated, giving very low digestibility values. Bandegan et al. (2010), determining standardized ileal amino acid digestibility in broilers, found the highest amino acid digestibility for dry-extruded expelled soybean, but lower values for poultry by-product meal and feather meal. In a follow-up study, Bandegan et al. (2011) determined standardized ileal amino acid digestibility in 21-d-old broilers given different samples of wheat, barley, peas, and flaxseed. Wheat was found to be more digestible than barley, and peas considerably more digestible than flaxseed. Flaxseed supported poor digestibility (40% for threonine to 67% for glutamate), Table 5. Variability of true protein and amino acid digestibility coefficients determined with cecectomized cockerels 1 Item Mean 2 CV (%) Protein Lysine Methionine Cysteine Threonine Wheat Corn Field pea Horse bean Full-fat soybean Toasted Extruded Cottonseed meal Rapeseed meal Rapeseed meal Sunflower meal Soybean meal Soybean meal 46/ Fish meal Meat and bone meal Feather meal (hydrolyzed) Blood meal From Rhône-Poulenc (1993). Mean digestibility. Glucosinolate >20 µg/g. Glucosinolate <20 µg/g.

8 and amino acid digestibility among samples was highly variable. Kim et al. (2012b) determined standardized ileal amino acid digestibility in 21-d-old boilers for soybean meal, canola meal, fish meal, and meat and bone meal. Soybean meal had a higher digestibility than canola meal, and overall, fish meal protein was more digestible than meat and bone meal. Rochell et al. (2013) determined standardized ileal amino acid digestibility using 30-d-old broilers, giving a comprehensive view of amino acid digestibility in meat and bone meal (10 samples). Digestibility coefficients were generally low, with mean amino acid digestibility ranging from 57% for cysteine to 77% for arginine. Digestibility was highly variable among the different samples. Processed Feedstuffs and Lysine Availability SYMPOSIUM: SUBSTRATES FOR EXOGENOUS ENZYMES 2407 For proteinaceous feedstuffs, whereby constituent amino acids may have undergone structural alteration either during processing (especially heating), conventional (after strong acid hydrolysis) amino acid compositional values and conventional true ileal amino acid digestibility values may be erroneous (Moughan, 2003; Moughan and Rutherfurd, 2008). Several amino acids (e.g., lysine, arginine, methionine, cysteine, and tryptophan) are susceptible to damage during processing, the best documented example of which is damage to the important dietary indispensable amino acid, lysine. Protein-bound lysine and free lysine, both having an epsilon amino group, can react with reducing sugars, fats, and their oxidation products, polyphenols and various dietary additives. The reaction between lysine and reducing sugars (the Maillard reaction), which takes place under mild conditions of processing, has been studied extensively. In the advanced stages of the Maillard reaction (brown pigment formation), lysine is completely altered and is not recoverable following hydrochloric acid hydrolysis during traditional amino acid analysis. In the early stages of the reaction, however, which are common under normal conditions of feedstuff processing, the deoxy-ketosyl derivative (Amadori compound) formed is hydrolyzed back to lysine in the presence of strong acids. Such reversion does not occur, however, under the milder conditions encountered in the bird s digestive tract. Because of this, heat-processing and unfavorable storage conditions can reduce dietary lysine in foods to a greater extent than is reflected by conventional amino acid analysis. Conventional amino acid analysis for these feedstuffs, overestimates the amounts of available lysine, and the same is also true for some other amino acids. Chemical assays for available lysine such as Carpenter s fluoro-dinitrobenzene (FDNB) lysine assay can be used, rather than conventional amino acid analysis, to provide estimates of chemically available (reactive) lysine in feedstuffs. The degree of overestimation can be considerable as highlighted by the data given in Table 6 for a range of meat and bone meals, where the FDNB available lysine is much lower than the total lysine as determined by conventional amino acid analysis. The data in Table 6 also highlight the considerable variation in available lysine in a byproduct such as meat and bone meal, and it is notable that in general the degree of overestimation of lysine declined as the amount of lysine increased. Not only is the estimated lysine content of a heatdamaged protein source subject to error from chemical determination, but the lysine content of ileal digesta is also subject to the same error of determination, meaning that conventional ileal lysine digestibility values determined for such feeds will be prone to error. It is also well established (Moughan et al., 1996; Moughan, 2003) that with damaged proteins, not all of the reactive lysine (chemically unaltered lysine molecules) is absorbed (reactive lysine may be only about 75% absorbed after early Maillard damage). Rather than focusing on the altered lysine molecules, their absorption and utilization, which was a focus of research in the 1970s and 1980s, it may be more productive to center on determining the uptake to the end of the small intestine of the reactive lysine molecules themselves, to give an estimate of available lysine (absorbed reactive lysine). A bioassay of lysine availability (true ileal digestibility of reactive lysine) has been developed (Moughan and Rutherfurd, 1996; Moughan, 2005) and applied (Table 7). Differences between traditionally determined true ileal digestible lysine and true ileal digestible reactive lysine (available lysine) can be considerable. To date the poultry industry has not taken advantage of this and similar bioassays, and there is much scope for their application. A similar scenario as for lysine also applies to the availability of methio- Table 6. Mean (n = 6) concentrations of total lysine and fluoro-dinitrobenzene (FDNB) reactive lysine in 12 samples of meat and bone meal 1 Sample Item Total lysine 2 (g/100 g) FDNB available lysine (g/100 g) % difference Adapted from Moughan et al. (1989), copyright 1989 John Wiley & Sons, Ltd. 2 After conventional acid hydrolysis. 3 Total available/total 100/1.

9 2408 Moughan et al. Table 7. Mean true ileal digestible lysine (conventional) content compared with true ileal digestible reactive (available) lysine True ileal digestible lysine 1 (g/kg) Item Conventional 2 Available 3 % Difference 4 Wheat Wheat Corn Corn Skim milk powder Cottonseed meal Whole milk powder Lactose-hydrolyzed milk powder Popped rice cereal product Grain-based cereal product Whole grain bread Heated skim milk powder Heated peas Soybean meal Split peas Determined in the growing pig or growing rat; from Rutherfurd and Moughan (2007). 2 Based on conventional amino acid analysis. 3 Based on determination of reactive lysine in diet and ileal digesta. 4 % Difference = [(conventional available/conventional) 100]; all differences were statistically significant (P < 0.01). nine and cysteine and that of other amino acids. The slope-ratio assay has been used in the past to determine amino acid bioavailability, but this approach is costly, time-consuming, produces data for 1 amino acid only per assay run, and the measure of availability is prone to having a high standard error of estimation. For these reasons, determining the uptake of the structurally unaltered amino acids using true ileal digestibility assays is attractive. Implications of Increased Dietary Undigestible Protein and Inaccuracy in Amino Acid Digestibility Coefficients (1) Higher Undigestible Dietary Protein. When feedstuffs of lower inherent quality (particularly those containing high amounts of plant fiber, non-starch polysaccharides, or ANF s) or where the protein quality has been impaired by processing, are used for diet formulation, there will be a higher undigestible protein component. This material passes into the hindgut of the bird, whereby it is subjected to fermentation by the bacteria in the colon and ceca. In such cases there will be a higher energy cost incurred by the bird for both fecal and urinary excretion (Birkett and de Lange, 2001a, b). Such higher metabolic costs may or may not be justified, dependent upon the costs of the respective feedstuffs. Additionally, when the proteinaceous material is fermented in the hindgut, ammonia, amines, and other compounds are produced that according to some may have toxic effects. There will be a greater environmental burden associated with the higher undigestible dietary component. Protein sources containing high amounts of undigestible protein often also contain factors that increase endogenous amino acid losses, and higher ileal undigestible DM leads to greater total endogenous protein losses. Thus a reduction in undigestible dietary protein is likely to have a marked effect on bird performance and on total nitrogen excretion. (2) Inaccuracy in Amino Acid Digestibility Coefficients. To allow for known errors in amino acid digestibility values and also to account for the variation in the digestibility (CV%) of an amino acid within a feedstuff, diet formulators often overformulate to give an insurance against undersupply. This is costly and poses an increased environmental burden. Also, amino acids supplied in excess of the bird s requirement must be oxidized, contributing disproportionately to dietary induced thermogenesis. This may have negative consequences at high environmental temperatures. Perhaps of greater consequence is when the application of inaccurate digestibility coefficients leads to an undersupply of the first-limiting amino acid relative to the amount required for optimal growth and maximum profits. Such inaccuracy, even leading to relatively small degrees of undersupply, can have significant negative effects on bird performance (Swennen et al., 2007) and financial returns. CONCLUSIONS It is important, both for meeting targeted bird performance and for minimizing the nitrogen environmental burden, to be able to determine amino acid digestibility accurately and precisely. Much work has been undertaken over the last 2 decades to greatly improve the prediction of amino acid uptake, and thus the undigestible component, in broilers and laying hens. The undigestible amino acid component in feedstuffs commonly used in poultry production is highly variable both among feedstuffs and within a feedstuff. The undigestible component can be

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