Future of Amino Acid Evaluation in Poultry Nutrition

Supply of the right amount of nutritionally adequate feed ingredients to the animal is key for efficient production of animal products. Attaining the goal of supplying the right amount of nutritionally adequate feed ingredients calls for careful evaluation and good understanding of the bioavailability of nutrients and energy in each feed ingredient formulated into a mixed diet. Critical attention should be given to the dietary amino acids (AA), energy, and phosphorus as those components account for major cost of feed. A deficiency of essential AA, energy, and phosphorus will result in a reduction in performance. In addition, excess of these components in the pig diet above the requirements is excreted and subsequently can be a source of environmental pollution.

The assumed composition of a mixed diet for poultry are calculated from chemical analyses of individual ingredients. But because nutrients in different ingredients are not utilized with the same efficiency, information on nutrients utilized is important for meeting the nutritional needs of swine and poultry. Nutrients and energy bioavailability of feed ingredients represent the proportion of ingested dietary nutrients and energy that are absorbed in chemical forms that render them potentially suitable for metabolism and or tissue accretion. There are difficulties and inaccuracies in the direct determination of bioavailability and the use of relative availability. Thus, nutrient and energy digestibility are often used in feed formulation as a more practical way of assessing the quantities of nutrients and energy that are absorbed.

Feed ingredients that contain protein are formulated into diets to supply amino acids for meeting physiological needs of the bird. The efficient production of animal products requires correct amount of nutritionally adequate feedstuffs in formulated diets. Attainment of this requires an understanding of the digestion characteristics and utilization of amino acids in the feed ingredients. Digestion characteristics and utilization of amino acids in a particular feed ingredient are not the same for all birds and stages of growth. Thus, amino acid utilization in feed ingredients could be affected by the species of birds, and within species, by the stage of development. A deficiency of an indispensable amino acid will result in a reduction in performance. Conversely, an excess of these components above the requirements of the bird is excreted, which can be a source of environmental pollution. Several methods can be used to evaluate the capacity of a protein-containing feed ingredient to meet the needs of a bird. For proteins, these include: Protein Efficiency Ratio (PER), Relative PER (RPER), Net Protein Ratio (NPR), Relative NPR (RNPR), Protein Replacement Value (PRV), Biological Value (BV), and Net Protein Utilization (NPU). Because dietary protein supply amino acids to the bird, relative amino acid bioavailability using slope ratio methodology or digestibility of amino acids can be used to evaluate the capacity of a protein-containing feed ingredient to meet the amino acid needs of a bird.

In this short communication, various methods for evaluating the potential of a feed ingredient to supply amino acids in poultry nutrition and pertinent examples for a few domesticated avian species are described. Furthermore, the additivity of AA in mixed diets and the currencies for such are discussed. 

The quality of intact feed ingredient protein generally is usually appraised by the capacity of the balance of amino acids in the protein to satisfy physiological needs of the bird. The amounts of nitrogen from the feed ingredient that is used for maintenance and/or production reflect accurately the adequacy of the assortment of amino acids relative to the requirement of the bird. Protein Efficiency Ratio (PER) is calculated as body weight gain of bird per unit of protein consumed. This measures the capacity of the protein to support growth and makes no allowance for the intake protein that is used for maintenance. In spite of this criticism, the method is widely used due to its extreme simplicity. Table 1 has protein efficiency ratios in published studies determined with broiler chickens and ducks.

An allowance is made for the ise of test protein to satisfy maintenance requirements in Net Protein Ratio (NPR). This is estimated from the weight loss of a group of birds fed a proteinfree diet. A group of animals is fed the test protein diet and a record of their body weight gain is taken. A similar group of animals is fed a protein-free diet and their weight loss is measured. The body weight gain of the test protein group is added to the weight loss (in positive terms) of the protein-free group, and the total is divided by the protein consumed by the test protein group. The assumption is made that the protein required to prevent a weight loss in animals fed the protein-free diet is equivalent to that needed for maintenance. In cases of different protein intakes, weight gain response to protein intake is not linear but deflects downwards as protein intake increases. Thus, NPR falls as protein consumption increases and frequently overestimates the value of poor quality proteins.

Assessment of protein quality using Protein Replacement Value (PRV) compares the extent to which a given protein can replace another protein (a reference protein) in terms of supporting protein deposition or nitrogen balance. The numerical difference between the two nitrogen balance supported by the reference protein source and the test protein source expressed as a percentage of the higher nitrogen intake, which represents the extent to which the test protein fails to biologically match the reference protein. Biological Value (BV) of a dietary protein is the portion of digestible nitrogen that is retained in the body. By this definition, the amounts of nitrogen from the feed that is used for physiological needs of the bird is an accurate reflection of the adequacy of the profile of amino acids relative to amino acid profile requirement for maintenance and productive functions of the bird. Surpluses of absorbed amino acids over and above the adequate assortment are deaminated and the nitrogen is excreted via the urine.

The determination of Net Protein Utilization (NPU) involves feeding a group of birds the test protein, determining the protein retained from the difference between nitrogen intake and nitrogen voided in excreta and expressing the nitrogen retained as a proportion of the nitrogen intake. The method combines both the nitrogen digestibility and the biological value in a single index as the product of biological value and nitrogen digestibility. Net protein utilization in published studies with broiler chickens and ducks are presented in Table 1. 

Table 1. Protein efficiency ratio (PER) and net protein utilization (NPU) in canola meal (CM), Soybean meal (SBM), wheat, barley, complete feed (diet), and corn for broiler chickens and ducks

The AA composition of a feed ingredient can determined by chemical analysis (wet chemistry) AA of the feed. However, chemical analysis does not provide information on the amounts of amino acid in the feed ingredient that are available to an animal. This information is obtained through well designed relative bioavailability of AA studies or AA digestibility studies. 

Relative bioavailability of AA has been commonly estimated using slope-ratio assay that provide relative information on the capacity of a feedstuff to supply a specific limiting AA for promoting growth. This assay is usually considered the ultimate standard against which other methods are judged due to the inherent feature of taking into account all of the components such as digestion, absorption and utilization that can affect bioavailability (Lewis and Bayley, 1995). The procedure and general concern of slope-ratio assay for pigs was well described by Batterham (1992). In this assay, a dose-response relationship between either crystalline form (Standard response relationship) or test AA source of interest and response criteria is established. Comparison between relationships is then conducted. This analysis tests for linearity of the slopes and lack of curvature – essential requirements for statistical validity; and for intersection of responses to standard (crystalline or synthetic) and test diets at the basal response (blanks) – essential requirements for fundamental validity. This assay is tedious, expensive, and limited in that only one AA can be assessed at a time. In addition, this has relatively high standard error and gives relative values only.

In the assay, a basal diet that is adequate in all other nutrients but deficient in the amino acid of interest is formulated. Graded levels of the crystalline form of the AA of interest are added to the basal diet for standard (reference) diets. A dose-response relationship between the crystalline AA and response criteria (weight gain and/or feed efficiency) is established. Graded levels of the amino acid of interest from the test protein are added to the basal diet for test diets and a dose-response relationship between the test protein and response criteria (weight gain and/or feed efficiency) is established. Assumptions are made that the dose-response relationships are linear (referred to as statistical validity) and lack curvature, and that the regression lines fitted to the standard and test protein intersect at basal response (referred to as fundamental validity). Estimates of bioavailability are only meaningful if the assumptions hold. Lack of curvature would be indicative of absence of stimulatory or depressive factors in the standard and test proteins, and intersection of the regression lines fitted to the standard and test protein at basal response would be indicative of response to test amino acid only and would therefore have common intercept. This assay is tedious, expensive, and limited in that only one AA can be assessed at a time. In addition, this has relatively high standard error and gives relative values only. Examples of the results of studies conducted to determine the relative bioavailability of AA in feed ingredients for broiler chickens and ducks are in Parsons (1986) and Adeola (1998). 

Due to the aforementioned disadvantages, slope-ratio method is not practical and applicable for all AA in all feed ingredients and diets for pigs. Therefore, digestibility of AA that is more suitable for evaluating dietary AA utilization in feed ingredients. Digestibility of AA represents portion of total dietary AA that is enzymatically hydrolyzed, fermented by digestive tract microbes and absorbed from gastrointestinal lumen.

Ileal digesta in birds can be collected either directly from the ileum after euthanasia or through an intestinal cannula. The most commonly used ileal digestibility method is the euthanasia method. With this method, birds are fed a marked experimental diet for 4 to 7 days after which they are euthanized and ileal digesta are collected. The ileum is defined as the section of the gastrointestinal tract extending from the Meckel's diverticulum to a point 20 mm cranial to the ileo-cecal junction. Although the majority of AA are absorbed prior to ileum, the distal half of ileum is the preferred site for ileal digesta collection to ensure marker recovery and complete absorption (Kadim and Moughan, 1997). The distal two-thirds of the ileum is considered the most accurate for sampling (Rodehutscord et al., 2012) and pooling of collected ileal digesta from 8 to 15 birds in the same cage to obtain sufficient ileal digesta for sample analyses.

The net disappearance of ingested dietary AA from the digestive tract proximal to the distal ileum is apparent ileal digestibility (AID) of AA. With this definition, AID of AA inherently does not differentiate between AA of undigested dietary and endogenous origin AA in the outflow at the distal ileum. Therefore, the level of crude protein in the test diet affects the apparent ileal digestibility. With low-protein diet, the relative contribution of endogenous AA to total AA in ileal outflow is high and as the CP level in the diet increases, the relative contribution decreases, therefore a primary concern with the use of AID in diet formulation is lack of additivity of AID in feed ingredients mixture that has low-protein feed ingredient.

Ileal endogenous AA consists of protein from gastric, pancreatic, and biliary secretions, and sloughed off mucosal cells. These endogenous AA losses are affected by protease inhibitors (e.g. trypsin inhibitor) in feed, fat, dietary fiber level, pectin, and level of dietary protein. There are basal, specific, and total (basal PLUS specific) endogenous AA losses. The use of uncorrected ileal digesta to calculate digestibility gives AID of AA. The correction of ileal digesta AA for total ileal endogenous AA losses gives true ileal digestibility (TID) of AA. If ileal digesta AA is corrected for basal ileal endogenous AA, standardized ileal digestibility (SID) of AA is obtained. Due to the arduous experimental approach in the determination of total ileal endogenous AA losses, the determination of basal ileal endogenous AA losses is more common using a nitrogen-free diet. The SID of AA is obtained by correcting ileal digesta AA for basal ileal endogenous AA losses determined from feeding a nitrogen-free diet. The SID of AA in soybean meal and canola acquired in recent studies are presented in Table 1.

Because there is no routine procedure for estimating specific ileal endogenous AA losses, it may be possible to calculate the specific losses by estimating the total (specific plus basal) endogenous losses and then subtract the basal losses from total losses. Procedures used to estimate total endogenous losses include the homoarginine technique, the feeding enzymehydrolyzed protein, and the isotope-dilution technique. Those of methods for estimating total endogenous losses are laborious, expensive, and require specialized equipment. As a consequence, estimates for total endogenous losses are not routinely determined for feed ingredient evaluation (Stein et al., 2007).

Direct methods, where individual feed ingredient was the only source of dietary AA, were used to evaluate AID and SID of AA in several feed ingredients in broilers, turkey poults, laying hens, and intact and cecectomized roosters (Huang et al., 2006, 2007; Adedokun et al., 2008; Adedokun et al., 2009). Roosters had higher AID of AA (for most of the AA) in corn and sorghum relative to values from laying hens. The AID of individual AA in wheat, corn, and sorghum, were higher in broilers than in laying hens and roosters (Huang et al., 2006). Laying hens had higher AID of AA in soybean meal than broilers and roosters (Huang et al., 2006). However, no significant difference was observed between broilers, laying hens, and roosters for soybean meal (Adedokun et al., 2009). The broilers used for the study by Adedokun et al. (2009) were 21 days old whereas they were 42 days old in the study by Huang et al. (2006). The AID of AA between 5- and 21-day old broiler chicks and turkey poults showed higher digestibility values for broilers for most of the AA (Adedokun et al., 2008). However, the difference between these values for most AA almost disappeared post standardization, especially on d 5, due to significantly higher ileal endogenous AA losses in poults relative to that of chicks (Adedokun et al., 2008). In another recent study with broiler chickens and ducks, Kong and Adeola (2013) observed differences in digestibilites of some indispensable amino acids between broiler chickens and ducks (Table 2).

There are reports of apparent and standardized ileal AA digestibility of several feed ingredients in broilers, turkey poults, laying hens, and roosters (intact and cecectomized) using the direct method where individual feed ingredient was the only source of dietary AA (Huang et al., 2006, 2007; Adedokun et al., 2008; Adedokun et al., 2009). Apparent ileal digestibility of individual AA in wheat, corn, and sorghum, were higher in broilers that in laying hens and roosters (Huang et al., 2006, Table 2). Roosters had higher ileal AA digestibility (for most of the AA) in corn and sorghum relative to values from laying hens. Ileal AA digestibility of soybean meal in laying hens was superior to those of broilers and roosters in the study reported by Huang et al. (2006). However, no significant difference was observed between broilers, laying hens, and roosters for soybean meal (Adedokun et al., 2009; Table 3). The broilers used for the study by Adedokun et al. (2009) were 21 d-old while they were 42 d-old in the study by Huang et al. (2006). There was no difference in apparent ileal AA digestibility of wheat between laying hens and roosters but AA digestibility of canola meal and cottonseed meal among the 3 strains of poultry were not different (Huang et al., 2006, Table 2). For corn, the digestibility for most of the AA was higher in broilers relative to that of the laying hens (Huang et al., 2007) but canola meal showed the highest variability in ileal AA digestibility between laying hens and broilers. Relative values of apparent ileal AA digestibility between 5- and 21- d-old broiler chicks and turkey poults showed higher digestibility values for broilers for most of the AA. However, the difference between these values for most AA almost disappeared post standardization (Table 3), especially on d 5, due to significantly higher ileal endogenous AA losses in poults relative to that of chicks (Adedokun et al., 2008). 

Table 2. Comparative standardized ileal digestibility of indispensable amino acids in soybean and canola meals for broiler chickens and ducks (Kong and Adeola, 2013b)

The linear regression approach has been suggested as one of the methods to estimate available AA in feed ingredients and diets for poultry (Short et al, 1999; Kluth and Rodehutscord, 2006). In this method, three or more diets containing graded levels of test ingredient are fed and ileal digesta collected from birds. A subsequent regression of the amount of AA digested at the end of the ileum in grams/day on the intake of AA in grams/day gives a regression slope that represents an estimate of digestibility of AA in the ingredients (Rodehutscord et al., 2004). Amino acid intake is the product of dietary AA concentration and feed intake. The digested amount of AA is calculated as the product of the daily AA intake of each test diet and the apparent ileal AA digestibility in the corresponding diet (Kluth and Rodehutscord, 2006, Kong and Adeola, 2013b). This method is based on the premise that the increment in the amount of digested AA as a function of their incremental intake is affected by all the ingredient-specific factors such as its digestibility as well as ability to induce specific endogenous AA losses (Rodehutscord et al., 2004). In this method therefore, the determination of basal endogenous AA losses, which is usually conducted separately and results in variation, is not necessary. 

Table 3. Apparent and standardized ileal amino acid digestibility in 5- and 21-d-old broiler chicks and and turkey poults (Adedokun et al., 2008) ¹

Kluth and Rodehutscord (2006) compared regression-derived estimates of ileal AA digestibility in soybean meal (SBM) and rapeseed meal (RSM) for broiler chickens, turkeys, and ducks. Digestibilities were not significantly different between SBM and RSM for broilers and turkeys. In ducks, digestibilities were lower than in broiler chickens or turkeys, and differences between SBM and RSM were detected for some AA in ducks. The authors concluded that differences among species cannot be explained by differences in basal endogenous AA losses among species and that amino acid digestibilities determined with broilers should not be used in formulating feed for ducks. Regression-derived estimates of ileal AA digestibility in SBM for broiler chickens and ducks in a recent study were reported by Kong and Adeola (2013). The ileal digestibility of AA from the test ingredients was assessed by multiple linear regression analysis using data on daily apparent ileal digestible AA and total AA intakes (See Figure 1 for examples on Lys and Met). The results showed differences between broiler chickens and ducks and that data obtained from broiler chickens should not be used to formulate diets for ducks. 

Additivity of digestible nutrients is a fundamental assumption when diets are formulated to meet animals’ requirements for nutrients. It is assumed that the amount of digestible nutrients in diet is equal to the sum of the digestible nutrients for each ingredient the mixed diet. Studies have been conducted to test the additivity of energy and nutrient digestibility in non-ruminant animals. Hong et al. (2002) reported that for White Pekin ducks, the true metabolizable energy and nitrogen-corrected true metabolizable energy for corn, soybean meal, and wheat red dog are all additive in complete diets. Fang et al. (2007) tested the additivity of true P digestibility (TPD) using substitution method and concluded the TPD values were additive. However, it is noteworthy to point out the apparent P digestibility values in some ingredients based on substitution method were highly variable (Fan and Sauer, 2002). The additivity in the context of P should be conceptualized as the representation of the true digestible P in the diet by the sum of digestible P from individual ingredient contributing P to the diet. More recently, Zhai and Adeola (2013) determined the regression-derived TPD in corn and soybean meal to be 40.5% and 36%, respectively for pigs. The expected TPD of 37.9% in a corn-soybean meal mixture, calculated from the determined TPD of corn and SBM, was not different from the determined of 37.5%. This indicated that true total-tract digestibility of P in corn and soybean meal for pigs are additive in corn-soybean meal diet.

For amino acids, it appears that AID for complete diet, predicted from AID of ingredients, underestimates AID for AA and CP in diets for growing pigs (Fan et al., 1993; Mosenthin et al., 2000; Stein et al., 2005). This underestimation could be a result of relatively higher contribution of basal endogenous loss (BEL) to the total ileal AA flow for low-protein ingredients as cereal grains (Rademacher et al., 2001). For this reason, some studies suggested that SID of CP and AA for ingredients should be more additive than AID in mixed diets because it is independent of BEL (Mosenthin et al., 2000; Stein et al., 2001). In recent studies with broiler chickens and ducks, Kong and Adeola (2013) reported that SID of AA in corn and soybean meal are more likely to be additive in a corn-soybean meal diet than AID values (Figure 2). 

Figure 1. Regression-derived ileal digestibility of lysine (LYS) and methionine (MET) for ducks (open circles) and broiler chickens (X) in soybean meal (Kong and Adeola, 2013).

 

Figure 2. Additivity and associative effect of apparent (AID) and standardized (SID) ileal amino acid digestibility (%) in corn-soybean meal-based diet for broiler chickens and White Pekin ducks (Kong and Adeola, 2013a).

Diets that that are formulated to more accurately match energy and nutrients requirements of poultry with dietary supply is central for sustainability of production. It is essential that evaluation of the capacity of feed ingredients to supply amino acids and requirements are expressed in the same currency because dietary supply and requirements are interdependent. Because dietary proteins are evaluated on the basis of capacity to meet the amino acid needs of the animal, methodology for determining digestibility estimates should be carefully selected. Although more laborious, regression method for estimating nutrient digestibility is robust and the determination of basal endogenous losses is not necessary. Standardized ileal digestibility of amino acids provides more accurate information for the formulation of diets than apparent ileal digestibility. An important advantage of using standardized ileal digestibility compared with apparent ileal digestibility is that values for standardized ileal digestibility are more likely to be additive in mixed diets for amino acids. 

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