Factors affecting endogenous amino acid flow in chickens and the need for consistency in methodology

Review Factors affecting endogenous amino acid flow in chickens and the need for consistency in methodology S. A. Adedokun,* O. Adeola,* C. M. Parsons,† M. S. Lilburn,‡ and T. J. Applegate*1 *Department of Animal Sciences, Purdue University, West Lafayette, IN 47907-2054; †Department of Animal Sciences, University of Illinois, Urbana 61801; and ‡Department of Animal Sciences, Ohio State University/OARDC, Wooster 44691 ABSTRACT Accurate estimation of ileal endogenous amino acid (EAA) losses is important when formulating diets on a standardized ileal digestible amino acid basis. In addition to the undigested and unabsorbed amino acids of dietary origin, amino acids of endogenous origin, which can be basal or diet specific, are found in digesta. The improvement in the techniques used in amino acid analysis as well as a shift from sampling excreta to ileal digesta has resulted in more accurate amino acid digestibility coefficients. Despite this, however, it is important to determine the amino acids in the digesta that are of endogenous origin. Although the need for standardization and its associated advantages is still subject to debate, it is important to evaluate how values from various methodologies compare. Several methods have been used to estimate ileal EAA flow. The classical methods, including the regression method, the use of nitrogen-free diet (NFD), and

the fasted cecectomized rooster method, are the most widely used. The criticisms with the last 2 methods are that birds are not in a normal physiological state and the ileal EAA flow is, therefore, underestimated. Different methods have resulted in different endogenous flow estimates, with the NFD method having the lowest values when compared with flows from the regression and highly digestible protein methods. In addition to the influence of methods on ileal EAA flows, the influence of the age of the birds on flow is important. Data on EAA losses are copious in the literature; however, variation in data across and within laboratories calls for investigation of factors contributing to the variation. This review compares results from different methods and examines the issue of repeatability and consistency of EAA losses data from different laboratories. Finally, composition of an NFD for estimating EAA losses is proposed.

Key words: amino acid, endogenous amino acid flow, nitrogen-free diet, poultry, standardization 2011 Poultry Science 90:1737–1748 doi:10.3382/ps.2010-01245

INTRODUCTION Competition between humans and livestock for grains and cereals is expected to increase as potential other uses for these feed ingredients are explored and as human population increases, especially in the developing world. This has resulted in an increase in the use of by- and coproducts that have lower amino acid digestibility. Additional reasons for the study of endogenous amino acid (EAA) losses in bird include the need to explore ways through which N is excreted into the environment and cutting the cost of production via a decrease in feed cost and an increase in birds’ uniformity. More than 20 amino acids have been identified and all are important for physiological functions in the ©2011 Poultry Science Association Inc. Received November 19, 2010. Accepted April 16, 2011. 1 Corresponding author: [email protected]

body (Ravindran and Bryden, 1999). Amino acids are the building blocks of protein but when in excess they are deaminated and the N arising from the process is excreted along with the unabsorbed N and amino acids of dietary and endogenous origin in the form of uric acid, ammonia, and urea (Goldstein and Skadhauge, 2000). The major N sources in excreta, according to Groot Koerkamp (1994), are uric acid (~70%) and undigested protein (~30%). In addition, amino acids of endogenous origin that are not reabsorbed contribute to N or amino acid excretion. Amino acids of dietary origin may not be available to birds as a result of several factors, including age of the bird, ingredient processing temperature, and techniques as seen in lysine, which may result in Maillard reaction, the presence of antinutritional factors (e.g., trypsin inhibitor), and level of dietary CP (Batal and Parsons, 2003; Fasina et al., 2004). The apparent amino acid digestibility coefficients for commonly available poultry feedstuffs have been

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documented (Ravindran et al., 2005). Apparent ileal amino acid digestibility is defined as the net disappearance of ingested dietary amino acid from the digestive tract proximal to the distal ileum (Stein et al., 2007). This definition, however, does not take into account the contribution of amino acids from bacteria that are resident in the gut because those amino acids are not truly of endogenous origin. Therefore, apparent amino acid digestibility coefficients underestimate amino acid digestibility. In the literature, the terms “true” and “standardized” ileal digestibility have been used interchangeably even though they are different. The term “standardized digestibility” is used when apparent digestibility coefficients are corrected for basal EAA losses. The term “true digestibility” is achieved when standardized digestibility is corrected for diet-specific as well as basal EAA losses (Lemme et al., 2004). This has been extensively discussed in the review by Stein et al. (2007). In this review, the term standardized digestibility will be used to represent apparent digestibility corrected for basal EAA losses. Endogenous amino acids contribute to the animal’s requirement, and information of EAA estimate could be used to standardized amino acid digestibility values. Diets are usually formulated to either meet or exceed nutrient requirements. Because not all nutrients in feedstuffs are available for growth and development of the animal, it is important to make a distinction between nutrient composition of a particular feed ingredient and nutrients that are available for biological purposes (bioavailable). Thus, amino acid digestibility in poultry is better explained on the basis of standardized amino acid digestibility (SAAD) values (Lemme et al., 2004) and hence the need to formulate diets based on digestible amino acid basis (Rostango et al., 1995; Dari et al., 2005). Several approaches have been used to estimate basal EAA flow (Moughan et al., 1990; Angkanaporn et al., 1994; Siriwan et al., 1994). The classical methods that have been used include the nitrogen-free diet (NFD; Kies et al., 1986; Furuya and Kaji, 1989), feeding of highly digestible protein (HDP; Furuya and Kaji, 1989; Chung, and Baker, 1992), and the regression method to estimate basal EAA flow. For a reliable SAAD value, results of EAA must be consistent and repeatable. Sources of EAA are numerous (Lien et al., 1997; Nyachoti et al., 1997; Montagne et al., 2004) and have been extensively reviewed (Nyachoti et al., 1997; Ravindran and Bryden, 1999; Lien et al., 2001; Moughan, 2003). Despite extensive work on basal EAA flow estimation, the need exists for a consensus diet and protocol so as to minimize variation across labs and the information within the scientific literature. Traditionally, amino acid requirements have been met using the total amino acid (TAA) content of each ingredient rather than on digestible amino acid basis. Diet formulation on a TAA basis together with the minimum CP levels often leads to excess protein intake and increased N and amino acid excretion. To minimize

nutrient excretion from poultry production and to improve uniformity in growth (during different production cycles and when different feed ingredients are fed), it is essential to refine the basis for feed formulation. The results from EAA flow estimation studies have been plagued with inconsistencies that have been attributed to the method of estimation, age, species and strain of birds, ileal collection method, choice of marker, and chemical analysis techniques (Ravindran and Hendriks, 2004; Adedokun et al., 2007a). Therefore, a consensus protocol and diet could go a long way toward minimizing the variability associated with EAA loss estimates for poultry species. In swine, Stein et al. (2007) attempted to unify the terminologies that are currently used to represent amino acid digestibility and bioavailability as well as proposed diets that can be used for EAA flow estimation. The objective of this review is to summarize the existing literature relative to factors affecting EAA flow in poultry. Additionally, the causes of inconsistency in the body of information available on EAA flow determination in poultry are reviewed.

EAA Amino acids of endogenous origin can originate from sources such as digestive secretions (saliva, bile, gastric, and pancreatic secretions as well as intestinal secretions), mucoproteins, sloughed intestinal epithelial cells, serum albumin, and amides (Hee et al., 1988; Nyachoti et al., 1997; Ravindran and Bryden, 1999). Although all amino acids are represented in the EAA, glutamic and aspartic acids, serine, and threonine are predominant in EAA (Chung and Baker, 1992; Adedokun et al., 2007b), with the 4 amino acids representing 34% (d 5) and 33% (d 21) of TAA flow in the broiler fed an NFD (Adedokun et al., 2007b). Endogenous amino acid losses can be divided into 2 major fractions (Nyachoti et al., 1997; Jansman et al., 2002; Stein et al., 2007). The first division is the basal (or diet-independent) endogenous flow representing the amino acids that are lost irrespective of whether the animal is fed. This remains constant and is not affected by dietary amino acid level. The specific (or diet-dependent) fraction arises as a result of the diet that is within the gastrointestinal tract (GIT) and the nature of the diet can influence the quantity of amino acids that are secreted into the GIT. Ileal amino acid digestibility coefficients that have been corrected for both basal and diet-specific EAA have been determined using the regression method (Rodehutscord et al., 2004; Kluth and Rodehutscord, 2006). Because this method requires feeding graded levels of the test ingredient, it invariably corrects not only the basal but also the ingredient-specific EAA losses. When apparent ileal amino acid digestibility values for soybean meal (300 g/kg of diet) from Rodehutscord et al. (2004) were corrected for EAA losses by apply-

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ing basal EAA losses from Adedokun et al. (2007b) and compared with values generated by the regression method (Table 1 from Rodehutscord et al., 2004), the difference in SAAD digestibility ranged between −1.5 and 7.3 percentage units. The lowest (0.4) and highest (7.3) percentage unit differences were for cysteine and aspartic acid, respectively (Table 1). Thus, the difference (although not statistically analyzed) is not dramatically different. The costs of the linear regression method (depending on how many concentrations of the ingredient are used) may make the single semipurified diet and correction for basal EAA losses a preferred approach. Whereas the regression method as described by Rodehutscord et al. (2004) results in a digestibility coefficient that is closer to “true” values, using the single semipurified diet (e.g., NFD) corrects only for basal EAA flow and therefore represents “standardized” values. Both methods, however, presume additivity when formulating a complete ration. The level of EAA flow depends on the presence of antinutritive factors (e.g., nature of dietary fiber and level of amino acid or protein intake; Sauer et al., 1977; Schulze et al., 1995). The separation of EAA from amino acids of dietary origin has been made possible with the development of the isotope dilution method (Souffrant et al., 1986). Basal losses have been measured using different techniques and the results are often inconsistent. The reasons for this may include the presence (or lack) of amino acids in the diet, the level of amino acid intake, the age of birds, or the species and strain of poultry. In addition to these potential sources of variability, it is still important to evaluate techniques that might be used to increase the reliability of data that are generated. A large body of data on EAA flow is required and will afford decision makers (nutritionists)

the opportunity to make decisions based on empirical data. The importance of determining EAA flow is that these values can be used to standardize digestibility coefficients of feed ingredients that can ultimately be used to formulate diets based on a digestible amino acid basis. An in-depth review of N cycling including endogenous N flow in the gut was written by Fuller and Reeds (1998). Mucin is a polymeric glycoprotein that comprises the main component of the mucus layer that covers the entire epithelium of the GIT (Montagne et al., 2004; Smirnov et al., 2006). Mucin comprises 5 monosaccharides (N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose, and sialic acids) and can be chemically classified into neutral or acidic subtypes (Montagne et al., 2004). Because mucin is constantly secreted and renewed and a high proportion of mucin remains undigested in the distal portion of the small intestine (Hoskins, 1984), ileal digestibility values (apparent) of some amino acids are compromised. The high concentration of threonine in ileal digesta has been attributed to the contribution of mucin to the ileal digesta and this may contribute to the low ileal apparent digestibility coefficients for threonine in many feedstuffs (Sauer and Ozimek, 1986; Lien et al., 1997). This is supported by data from our laboratory (Adedokun et al., 2007b,c; Horn et al., 2009). Lien et al. (2001) reviewed in details the potential dietary effects on mucin. Four amino acids (proline, glycine, glutamic acid, and aspartic acid) were reported by Taverner et al. (1981) to be the predominant amino acids in the ileal digesta of pigs. In chickens, however, glutamic acid, aspartic acid, serine, and threonine were the prominent amino acids (Siriwan et al., 1994; Angkanaporn et al., 1997; Adedokun et al., 2007b,c; Golian et al., 2008).

Table 1. Comparison of standardized ileal amino acid digestibility of soybean meal in 21-d-old broiler chickens Item Essential amino acid  Arginine  Isoleucine  Leucine  Lysine  Methionine  Phenylalanine  Threonine  Valine  Average Nonessential amino acid  Alanine   Aspartic acid  Cysteine   Glutamic acid  Glycine  Serine  Average

Standardized via nitrogen-free diet1

Standardized via regression2

Difference

  85.1 80.0 81.0 87.2 90.2 83.5 79.4 78.9     76.1 75.4 80.3 90.3 75.3 81.9  

  84 77 78 84 87 85 78 77     75 75 73 85 72 79  

  1.1 3.0 3.0 3.2 3.2 −1.5 1.4 1.9 2.3   1.1 0.4 7.3 5.3 3.3 2.9 3.4

1Endogenous correction for apparent ileal amino acid digestibility for soybean meal (300 g/kg of diet) from Rodehutscord et al. (2004) using basal endogenous amino acid flow from broilers fed a nitrogen-free diet (Adedokun et al., 2007b). 2Standardized amino acid digestibility values from regression of 0, 300, and 600 g of soybean meal/kg of diet (Rodehutscord et al., 2004).

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Threonine, serine, and proline represent 28 to 33, 13 to 16, and 7 to 24%, respectively, of the TAA content of mucin (Lien et al., 2001). Across species (rats, pigs, and poultry), type of poultry (broilers, roosters, and laying hens), and methodology [NFD, HDP, enzyme hydrolyzed casein (EHC), and guanidination methods], the predominant EAA found in the ileal digesta are glutamic acid, aspartic acid, threonine, serine, glycine, leucine, and proline (Hodgkinson et al., 2003; Ravindran and Hendriks, 2004; Adedokun et al., 2007a; Table 2). These findings suggest that neither the method of estimating EAA flow nor the concentration of dietary amino acid influence the order of predominant amino acids of endogenous origin rather the concentration of EAA in the digesta. Therefore, the relative contribution of amino acids of endogenous origin from different sources is influenced by the diet. Furthermore, these amino acids are high in the endogenous secretions of germ-free chicks (Salter and Fulford, 1974). Threonine, serine, proline, and valine have also been reported by Lien et al. (1997) to be high in mucin. To fully understand the effect of mucin on amino acid digestibility, evaluation of the effect of dietary components on the different monosaccharides that make up the mucin components is needed.

FACTORS AFFECTING ENDOGENOUS AMINO ACID FLOW Mucin Turnover Rate A considerable amount of mucin is digested in the hind gut (Hoskins, 1984) despite the fact that mucins are mostly mucoproteins that are protected from further proteolysis in the small intestine by a coat of oligosaccharides (Hoskins, 1984; Lien et al., 1997). Smirnov

et al. (2004) reported that mucin protein content increased distally along the small intestine of the chicken and intestinal mucin is continually degraded and renewed (Montagne et al., 2004; Smirnov et al., 2004). Because of the dynamic nature of mucin degradation and synthesis, the turnover rate is not regular; therefore the thickness of the mucus layer may vary depending on the balance between synthesis and degradation. This balance can be influenced by the nature of the diet or presence or virulence status of pathogens (Sharma et al., 1997; Montagne et al., 2004). The concentration of amino acids in the diets may also influence this balance. Throughout the small intestine in chicks the mucus layer is similar in thickness (Smirnov et al., 2004), whereas in the rat the layer is similar in the duodenum and jejunum and thicker in the ileum (Atuma et al., 2001). When 4-wk-old broiler chicks were deprived of feed and water for 72 h, the intestinal surface area was reduced along with a thinner mucus adherent layer (Smirnov et al., 2004). Similar observations were observed in laying hens starved for 36 h (Yamauchi et al., 1996) and in rats fed parenterally (Sakamoto et al., 2000). This suggests that the presence of feed in the GIT is important for the continual synthesis and secretion of mucus. The most obvious effect of starvation in 28-d-old broiler chicks was the enlargement of the goblet cells in the small intestine, in addition to decreased mucin thickness and increase in mucin protein concentration (Smirnov et al., 2004). The increase in goblet cell size may be an indication that the GIT sensed the need to increase mucin production. The role of feed withdrawal in intestinal physiology and mucus secretion was reported by Thompson and Applegate (2006). They reported a 46% decrease in mucus from 0 to 24 h of feed withdrawal. This observation underscores the importance of DM intake or changes in

Table 2. Endogenous amino acid flow (mg/kg of DM intake) in broilers fed nitrogen-free diets Adedokun et al., 2007b

Item Age, d Essential amino acid  Arginine  Histidine  Isoleucine  Leucine  Lysine  Methionine  Phenylalanine  Threonine  Tryptophan  Valine Nonessential amino acid  Alanine   Aspartic acid  Cystine   Glutamic acid  Glycine  Proline  Serine  Tyrosine   Total amino acid 1NA:

data not available.

Golian et al., 2008

Ravindran et al., 2004

Soleimani et al., 2010

Siriwan et al., 1993

21   168 73 162 251 181 50 154 274   214

21   230 91 200 298 173 65 420 434 71 270

35   280 158 287 439 209 101 287 512 95 417

42   469 203 327 535 383 81 394 538 50 398

    230 100 390 490 220 70 250 450 NA1 370

177 340 136 420 205 240 260 124 3,952

217 430 143 492 245 289 343 — 4,368

293 607 262 721 508   424 253 5,817

349 574 103 851 400 341 547 265 6,810

250 490 NA 630 280 NA 390 660 NA

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nutrient status of the bird on intestinal mucin turnover and gut integrity. The problem with determining EAA contribution from mucin and thus the aforementioned issues related to level of differences across the literature are likely attributable to methodologies used with the bioassay itself. Notably, several physiological factors influence the level and degradation rate of mucins.

Dietary Protein or Amino Acid The concentration of dietary protein or amino acids is another factor that may influence EAA losses via increased digestive enzyme secretion and mucin dynamics in the gut of nonruminant animals. In the work of Montagne et al. (2000), preruminant calves were fed milk substitutes with CP ranging from 140 to 278 g/kg and mucin flow at the duodenum significantly increased with increasing dietary CP. One likely explanation is increased mucus hydrolysis leading to increased mucin concentration in the digesta. Additionally, the increase in EAA losses could be attributed to the increased secretion of digestive enzymes in response to increased protein and peptides in the gut. This may be responsible for the high EAA flow observed with increasing level of intact casein as reported by Adedokun et al. (2007b,c) and Golian et al. (2008) or EHC or guanidinated casein (Hodgkinson et al., 2003; Ravindran et al., 2004) compared with when NFD was fed. Furthermore, it has been reported that the addition of plant protein (raw pea flour) to milk substitutes increases mucin flow in calves (Montagne et al., 2000).

Dietary Fiber Dietary fiber is one of the feed fractions that has the ability to affect mucin dynamics, including mucin excretion, in the GIT of animals. A combination of increased fiber (wheat bran) added to an NFD and high DM intake resulted in significant increase in mucin output in the digesta (Nyachoti et al., 1997; Montagne et al., 2003). Sauer et al. (1977) also reported an increase in EAA flow in pigs fed NFD with increased dietary fiber. Kluth and Rodehutscord (2009) concluded that a high fiber (cellulose) concentration in the diet of broiler chickens increases the EAA loss, but the composition of the EAA fraction was not affected. Solubility of the fiber as well as the water-holding capacity of the diet, especially the capacity of dietary fiber to absorb water, is important to overall EAA flow (Leterme et al., 1996, 1998). Additionally, dietary fiber can affect mucin composition. The role of short-chain fatty acids formed in the large intestine via fermentation can also influence mucin dynamics (Barcelo et al., 2000). Hence, the level and nature of dietary fiber may explain some of the increases in the EAA flow in nonruminant animals (Montagne et al., 2004) as well as inconsistencies in EAA flow observed in different studies. Using the homoarginine technique, Angkanaporn et al. (1994) reported that high concentration of wheat

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pentosan (35 g of pentosan/kg of diet) resulted in a decrease in amino acid digestibility and an increase in EAA flow. The addition of insoluble fiber (purified cellulose or powdered polyethylene) at higher concentration (91.5 g/kg of diet) did not significantly affect apparent digestibility. Studies using adult cockerels fed an NFD containing potato starch–pectin resulted in increased EAA secretions compared with birds fed NFD with insoluble fiber (cellulose; Parsons et al., 1983; Parsons, 1984). Dietary fiber fed to rats also increased endogenous secretions, presumably because of the abrasive action of the fiber on the mucosal surface of the gut (Beames and Eggum, 1981). It appears that the presence of antinutritive factors in poultry diets will reduce amino acid digestibility and increase EAA secretions (Angkanaporn et al., 1994). Tanins, lectins, and protease inhibitors have also been shown to increase EAA production (Nyachoti et al., 1997). The presence of antinutritive factors in the diet stimulates digestive enzymes secretion into the gut, increases sloughing of epithelial cells, and increases mucin production, resulting in reduced apparent amino acid digestibility (Montagne et al., 2004).

Phytate Phytic acid [inositol hexaphosphate, myoinositol 1,2,3,4,5,6-hexakis(dihydrogen phosphate)], a naturally occurring phytochemical, is found in almost all plant feedstuffs (Graf, 1983). The ability of phytate to form complexes with proteins and other nutrients is thought to be a mechanisms through which phytates exert antinutritional effects. Phytate can also inhibit the activity of trypsin by binding with calcium and inhibiting the conversion of trypsinogen to trypsin (Madhav and Krikorian, 1982). Onyango et al. (2009) reported that phytic acid increases mucin and EAA losses from the GIT of chickens and that the extent of the loss is a function of the type of phytate in the diet. The likelihood of negative effects of phytate on protein utilization was suggested by Rojas and Scott (1969), whereas Kies et al. (2001) proposed a mechanism by which this might occur. The negative effects of phytate on protein digestibility could be attributed to the de novo formation of protein–phytate complex in the GIT (Kies et al., 2001). These complexes have been reported to be resistant to pepsin activity (Selle et al., 2000). Furthermore, Ravindran et al. (1999) and Selle et al. (2006) proposed that the decreased apparent amino acid digestibility values observed in grains and cereals may be a result of phytate-induced increases in EAA flow. It was reported in broilers that phytates (as sodium– or magnesium–potassium–phytate) significantly increased TAA excretion by about 28% whereas the addition of phytase reduced this increase in EAA loss by about 20% (Cowieson et al., 2004; Onyango et al., 2009). The increased EAA flow was attributed to increased secretion of mucoproteins as a result of phytate stimulation of the GIT (Cowieson et al., 2004).

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Type of Index Marker The variability that has been reported in some digestibility studies as well as in estimating EAA losses could be attributed to the type of index marker included in the diet. Although the marker itself likely has no bearing on EAA flow, passage of the marker, segregation, and analytical variation of different markers may contribute to differences between published values. The magnitude of the variations in amino acid digestibility may be associated with the type of index marker used as reported in the metaanalysis by Selle et al. (2006), with titanium oxide and acid insoluble ash showing a larger increase in amino acid digestibility with the addition of phytase in the diets when compared with diets containing chromic oxide as the inert marker. Oberleas et al. (1990) challenged the validity of the assumption that chromic oxide is uniformly distributed in the transit rate of digesta and the marker is uniform. This observation, however, must be subjected to further empirical test, especially with all the markers being tested in the same study and across similar ages.

Gut Health The GIT health status of the bird is yet another important factor that may contribute to EAA loss. Because most of the studies that have looked at EAA losses in poultry have been conducted using birds in cages, the birds are removed from exposure to potential bacteria and pathogens, which might influence gut health. Although it has not been directly determined, raising birds on litter could lead to an increase in bacterial challenge to the gut and potentially disrupting the intestinal mucus layer and increasing gut turnover, secretion, and passage rate. Regarding intestinal turnover, presence of intestinal microflora increases turnover rates of the duodenum (Cook and Bird, 1973) and ileum (Coates, 1980) by 1.6 and 2.6 fold, respectively. When pathogens are present, however, this turnover can increase quite dramatically and thus influence the endogenous amino acid flow. For example, Fernando and McCraw (1973) reported a 3.1, 1.9, and 1.9 times faster epithelial turnover in the duodenum, jejunum, and ileum when chicks were challenged with Eimeria acervulina (when turnover was measured 3 and 4 d postchallenge). Similarly, additional environmental pressure due to bacterial load can influence other aspects of endogenous amino acid losses through mucin production. For example, birds that are exposed to increased bacteria load in the environment (i.e., recycled litter) have higher requirements for threonine (Corzo et al., 2007). Because the mucin layer contains a diverse array of carbohydrate components, they contain a wealth of potential attachment sites for commensal and pathogenic bacteria (Sonnenburg et al., 2004). Mucin production is largely a balance of production, secretion, degradation, and sloughing. Notably, some pathogens secrete

proteases and glycosidases to utilize mucin as a nutrient source. Some pathogens will produce virulence factors that can increase mucin degradation (e.g., Yersinia enterocolitica and Helicobacter pylori), whereas others can produce toxins that increase mucin production and secretion (e.g., Vibrio cholerae; Moncada et al., 2003). In broilers, a causative link has been noted by Collier et al. (2008) wherein Clostridium perfringens actively induces mucin production and active mucolysis aiding in rapid proliferation, resulting in necrotic enteritis. The extent to which different environmental and bacterial stressors affect EAA flow, however, has largely not been quantified.

Age, Species, and Method Effects The effect of age on ileal EAA estimates has been reported for poultry (Ravindran and Hendriks, 2004; Adedokun et al., 2007a) and swine (Mariscal-Landín and Reis de Souza, 2006). In broiler chicks and turkey poults, Adedokun et al. (2007a,b) reported that ileal EAA (mg/kg of DM intake) flow in 5-d-old broiler chicks was approximately twice that observed on d 15 or 21 whereas the difference in flow during the same period in turkey poults was about 3-fold higher than that of broiler chicks. These results point to 2 things. First, under a given set of conditions, the concentration of amino acids of endogenous origin in the ileal digesta on d 5 could influence the apparent digestibility values of various ingredients. This brings into question the extent to which amino acid digestibility of feed ingredients increases after the first week of age. Additionally, the drastic reduction in ileal EAA flow on d 15 relative to d 5 (68% reduction for poults and 53% reduction for chicks, TAA) as well as the lack of significant difference in ileal EAA flow between d 15 and 21 is interesting. Nyachoti et al. (2000) attributed differences in endogenous N (EAA) flow observed in pigs to differences in the efficiency of endogenous N (EAA) reabsorption rather than to differences in the rate of protein synthesis in the gut. This means that correction for EAA loss during the first week may be crucial in reducing N excretion in poultry. The possibility that the effect of age on EAA flow might become less significant as a bird matures is supported by the results obtained by Ravindran and Hendriks (2004) when EHC (180 g of EHC/kg of diet) was fed to 6-wk-old broilers, 70-wk-old layers, and 70-wk-old roosters. This is also supported by the findings of Adedokun et al. (2007b,c). In swine, Hodgkinson et al. (2000) reported that ileal EAA flow in 6.4-kg piglets was higher than in 53-kg pigs as estimated using graded concentrations of EHC. This age effect is similar to that recently reported in broiler and turkey poults (Adedokun et al., 2007a,b). The effect of increasing the concentration of peptides (or protein) in the test diet also resulted in an increased EAA flow at the terminal ileum of birds (Ravindran et al., 2004; Adedokun et al., 2007a) and pigs (Hodgkinson et al., 2000). Mariscal-Landín and Reis de Souza

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(2006), however, reported no linear effect when 8, 16, 24, or 32 g of N (from casein) was fed to 6.4-kg piglets. In young poultry (chicks and poults), increasing the concentration of dietary casein (0, 5, 10, or 15%) resulted in a linear increase in EAA flow. The respective increases for TAA flow were 42, 49, or 59% (d 5) and 40, 55, or 72% (d 21) when compared with birds fed an NFD (Adedokun et al., 2007a). This observation is similar to what Hodgkinson et al. (2000) reported for pigs fed diets containing 5, 10, or 20% EHC. The N flow was 11, 39, or 70% more for the EHC treatments (5, 10, or 20%, respectively) when compared with flows from pigs fed an NFD. Ravindran et al. (2004) also reported that the presence of dietary protein (19% CP from EHC diet and 19% CP from guanidinated casein diet) resulted in 42% (EHC) and 63% (guanidinated casein) increase in N flow. For all of the amino acids, N, and CP, no difference in EAA flow was found between the EHC and guanidinated method; however, flow from birds fed these diets was higher than that of birds fed the NFD. In the studies reported by Adedokun et al. (2007b,c), basal EAA flow was also estimated by regressing ileal EAA flow against dietary casein concentration. The results showed that on d 5, basal ileal EAA flow obtained from the regression method was higher (chicks) or lower (poults) than flow from the NFD method for most of the amino acids whereas on d 21 (chicks and poults) no difference was found between the 2 methods. The similarity between the NFD and regression method calculated EAA flow on d 21 is in agreement with the findings of Golian et al. (2008). It is important to note that the concentration of dietary protein and amino acid, especially the minimum concentration fed, may have some effect on basal EAA flow estimation using the regression method. It is important to determine how significant such an effect could be in a single study. Amino acids of dietary origin elicited a response in EAA flow in pigs that was similar to that observed in poultry. Ileal EAA flow from 100-kg pigs fed a semipurified diet containing 16.71% intact casein was higher for most amino acids than from pigs fed an NFD (Chung and Baker, 1992). The results from the study by Hodgkinson et al. (2003) in 179-g rats and 19.2kg pigs showed that the guanidination and the isotope (15N) dilution methods gave a lower estimate of ileal EAA and ileal endogenous N flow, respectively, than the EHC method in both species. Also, the 15N infusion method gave a higher estimate of endogenous N flow than the EHC method in the pig. The large difference in EAA observed between NFD and semipurified diets containing intact casein, EHC, or guanidinated casein could be the result of 2 factors. One, the proportion of casein that was not completely digested or absorbed may have increased with increasing concentration of dietary protein. The undigested and unabsorbed protein of dietary origin has to be significant to justify the large difference observed. The alternative explanation is that an increasing in-

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take of dietary protein resulted in an increase in endogenous protein secretion into the gut (Adedokun et al., 2007b,c; Golian et al., 2008), which is a more plausible explanation. When results obtained from pigs fed graded concentrations of EHC (Hodgkinson et al., 2000) were compared with results from broiler chicks (Adedokun et al., 2007b), the casein in the HDP diet could be said to increase EAA secretion to a greater extent in broiler chicks relative to pigs. For example, the ileal EAA flow for threonine in pigs fed 5% EHC was 47% of the flow observed with 10% dietary EHC. In our study, threonine flow in diets containing 5% HDP was 88% (d 5) or 82% (d 21) of the flow observed with a diet containing 10% HDP (Adedokun et al., 2007b). Furthermore, N flow from pigs fed the 5% EHC diet was about 68% compared with flow from pigs fed the diet containing 10% EHC (Hodgkinson et al., 2000). This can be compared with 87% (d 5) or 74% (d 21) for TAA in the chick study (Adedokun et al., 2007b). Researchers have criticized this method (feeding of intact casein) because attempts were not made to differentiate between amino acids from casein and those from endogenous origin. Despite this, however, comparing results from the chick study (Adedokun et al., 2007b) with that from the pig study (Hodgkinson et al., 2000) suggests that the peptide and amino acids from the casein diets were highly digested and absorbed. Additionally, apparent ileal amino acid digestibility values of the casein diets were corrected for EAA flow using the NFD method. Interestingly, the standardized ileal amino acid digestibility values for the casein diets were more than 90% in most cases (Table 3). Furthermore, enzymatically hydrolyzed and intact casein has been shown to be completely digested by growing pigs and chicks (Parsons et al., 1982; Moughan and Smith, 1985; Kies et al., 1986) whereas Wang and Fuller (1989) reported that AA are completely absorbed in pigs fed a semipurified diet based on intact casein and crystalline amino acid. When using an EAA loss value for standardization, species and strain effects as well as age of birds should be taken into consideration. No difference was observed in EAA flow between 21-d-old broilers and 37-wk-old single comb White Leghorn laying hens fed an NFD or HDP diet. However, the EAA flow in 104-wk-old cecectomized roosters was higher (3.5- to 12-fold) than in broilers and laying hens (Adedokun et al., 2009). Moreover, no significant difference (NFD vs. HDP) was observed in the EAA flow in fasted and precision-fed cecectomized roosters, but the EAA in the fasted roosters was lower relative to cecectomized roosters fed NFD or HDP diets (Adedokun et al., 2009). On d 5, the method of standardization (NDF or HDP) did not have any significant effect on the SAAD of the plant source feed ingredients tested (Adedokun et al., 2008). However, the HDP method of correction in broiler chicks resulted in higher SAAD values on d 21 for light colored dried distillers grains with solubles, canola meal, corn, and

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Adedokun et al.

Table 3. Standardized ileal amino acid digestibility (%) in 21-d-old broiler chicks and turkey poults fed diets containing 3 levels of casein (50, 100, and 150 g/kg)1 Broiler chicks Item

50

Essential amino acid  Arginine  Histidine  Isoleucine  Leucine  Lysine  Methionine  Phenylalanine  Threonine  Tryptophan  Valine Nonessential amino acid  Alanine   Aspartic acid  Cystine   Glutamic acid  Glycine  Proline  Serine  Tyrosine

Turkey poults

100

150

SD

95.54 96.08 90.98 97.16 96.38 95.20 97.56 93.74 NA3 94.06

95.83 96.20 91.58 96.87 96.87 95.57 97.45 94.05 NA 94.48

96.40 96.32 91.74 97.04 96.32 95.56 97.76 92.96 NA 94.62

1.506 1.004 2.114 0.919 1.046 1.283 0.969 2.216 NA 1.509

92.72 93.20 75.26 93.56 88.92 96.94 87.18 96.96

92.87 93.48 78.62 93.88 90.20 97.02 87.00 97.27

92.92 93.24 82.70 93.52 91.18 96.72 87.22 97.46

1.972 1.873 7.511 1.508 3.113 0.919 3.253 1.123

Linear effect2

Linear effect2

50

100

150

SD

0.38 0.71 0.58 0.84 0.93 0.66 0.75 0.59 NA 0.79

100.03 99.12 94.25 99.22 99.10 98.10 100.40 102.13 NA 98.62

99.97 99.07 94.90 99.18 99.17 98.42 100.17 99.50 NA 97.72

97.82 97.62 93.46 97.88 97.66 97.46 98.64 96.16 NA 95.84

2.293 1.496 2.299 1.333 1.627 1.247 4.847 2.689 NA 2.067

0.13 0.12 0.58 0.12 0.17 0.41 0.04 0.002 NA 0.04

0.88 0.97 0.14 0.97 0.27 0.71 0.98 0.49

98.62 98.43 107.98 95.92 100.53 99.60 93.87 99.97

98.10 98.02 100.45 96.27 98.77 99.08 93.33 99.73

94.72 95.64 87.96 95.46 94.62 98.10 91.90 98.42

3.340 2.563 5.829 1.711 3.902 0.913 2.977 1.167

0.08 0.09 0.0001 0.67 0.03 0.02 0.29 0.05

1Endogenous correction was by nitrogen-free diet. There were 6 replicate cages/casein level. Each cage represents an experimental unit with 8 birds/ cage. Calculated using endogenous flow data reported by Adedokun et al. (2007b,c). 2Significant linear effect; P < 0.05. 3NA: data not available.

soybean meal, with an average difference value across amino acids of 3, 3, 7, and 2 percentage units, respectively (Adedokun et al., 2008).

REPEATABILITY AND CONSISTENCY OF ILEAL ENDOGENOUS AMINO ACID VALUES One major concern of incorporating digestible amino acid values into commercial formulations is the lack of repeatability or consistency in available ileal SAAD data. In fact, variability both within and between laboratories can be considerable (Donkoh and Moughan, 1999). Adedokun et al. (2007b,c) conducted studies using NFD and HDP diets to investigate the variability and repeatability issues associated with EAA estimation. This was done across 2 laboratories and locations for broiler chicks and turkey poults (Adedokun et al., 2007b,c). Experimental diets were made from the same batch of ingredients, mixed at the same time and at the same location, before being shipped to the respective locations where similar studies were conducted. Similar protocols (feeding, sampling, and sample processing) were used at the 2 locations and chemical analyses were conducted on digesta and diets at the same laboratory. Results from these studies showed that EAA estimates can be repeatable across laboratories as long as the variations associated with feed ingredients, diet composition and mixing, and sampling and processing methods as well as analytical methods were similar. Flows from the NFD were found to be the most consistent across laboratories based on the associated standard

errors (Adedokun et al., 2007b,c). Some reasons that can be adduced for the lack of consistency in previous data and reports may include variations in the batch of feed ingredients used; the proportion of the different components in the diet; the level of fiber; ileal content collection method (squeezing vs. flushing); the choice of marker; age, species, and strain of birds; level of casein (or CP content) of the diet; and variations associated with chemical analysis. For instance, the composition of the NFD used to generate data presented in Table 2 is different across citations. To overcome this problem, it may be necessary to use a consensus diet formulation to estimate EAA flow in poultry as recently suggested by Stein et al. (2007) for pigs. Therefore, an NFD for estimating ileal EAA losses in poultry is proposed (Table 4). To minimize some of this variation, the following are suggested:

1. Large number of birds (15) per cage for enough ileal samples. For birds to be sampled in the first 10 d, 2 replicate cages may need to be pooled. 2. Basal ileal EAA flow should always be determined in studies in which EAA flow correction would be made. 3. Digesta should not be squeezed but flushed with distilled water to minimize EAA from damage to the epithelial surface of the ileum. 4. Consistency in chemical analysis is important. Results of lysine analysis from different laboratories on the same feed samples was 5.7% compared with within-laboratory variation of 1.4% (American Association of Feed Control Officials

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REVIEW diet1

Table 4. Proposed dietary composition of the nitrogen-free to be used to estimate endogenous amino acid losses in poultry (g/kg, on as-fed basis) Ingredient

Value, g/kg

Corn starch Dextrose Fiber Soybean oil Vitamin–mineral premix2 Monocalcium phosphate Limestone NaHCO3 KCl K2CO3 MgO Choline chloride Inert marker3 Total

200.5 640.0 50.0 50.0 5.0 19.0 13.0 7.5 2.9 2.6 2.0 2.5 5.0 1,000

1Na+ + K+ − Cl− − milliequivalency value of 108. Sodium requirement for 0- to 3-wk-old broilers is 0.2% (NRC, 1994). 2Vitamins and minerals in the premix should be adequate for the age of the bird. 3The choice of index marker is important and may affect the final value.

Feed Check program, Victoria Siegel, personal communication). 5. A diet adequate in all nutrients should be fed before when the NFD would be introduced.

DIET ELECTROLYTE BALANCE Although the need to use NFD for basal ileal EAA losses has been advocated above, it is important to note that variations do exist between NFD that have been reported in the literature, especially as it relates to the diet electrolyte balance or milliequivalent (mEq) values of Na+ + K+ − Cl− per kilogram of diet. For instance, the Na+ + K+ − Cl− mEq values of NFD used were 219 (Adedokun et al., 2007a,b), 250 (Golian et al., 2008), and 372 (Adeola and Ileleji, 2009), respectively. The Na+ + K+ − Cl− mEq value in the NFD proposed in this review is 108. Based on our observation, birds raised on an NFD with mEq value of 219 produced excessively watery excreta, which may be a result of drinking of excess water compared with birds raised on NFD with mEq values of 130 and 108. However, an NFD with mEq value of 108 was proposed because requirements for Na+, K+, and Cl− were met. Hence, the need exists to develop a consensus protocol that will minimize variations in EAA losses as a result of differences in the relative proportions of these mineral elements. The role of Na+, K+, and Cl− ions in tissue protein synthesis, maintenance of homeostasis (internal and external), electric potential of cell membranes, and acid– base balance has been reported (Borges et al., 2004; Olanrewaju et al., 2007). Borges et al. (2003) reported that optimal dietary electrolyte balance for weight gain and feed conversion ratio was 236 and 207 mEq/kg, respectively, which is similar to about 250 found in a typical corn–soybean meal-based starter diet. Further-

more, we observed that values of 219 mEq of an NFD (dietary Na concentration = 0.6%) resulted in increased water intake and, therefore, watery excreta. However, when the mEq value was reduced to 130 (dietary Na concentration = 0.3%) less water consumption was observed (S. A. Adedokun, unpublished data). This observation can be explained by a lack of protein in the diet. This observation is supported by Mongin (1981) who reported that the nature of dietary protein has an effect on endogenous acid production. The effects of lack of protein in the NFD might be partly responsible for our observation when diets with 219 or 130 mEq/kg were fed. Therefore, it is necessary to make sure that the concentrations of Na+, K+, and Cl− ions in NFD meet and do not exceed their respective requirements at a given age.

CURRENT UNKNOWNS Despite the large volume of information available in the literature on EAA flow in poultry, many questions still need to be addressed, including the following: 1. The use of an NFD or a diet containing casein. Different levels of protein, peptide, and amino acids in a diet elicit different levels of EAA losses. It is therefore expected that the levels of casein or HDP in the diet may influence the resulting EAA loss estimate. It is expected that lower levels of casein or HDP in the diet would give EAA value that is closer to the real basal EAA value (when using the regression method). 2. Effect of Na+ and mEq value on EAA flow. In previous studies when the Na+ concentration in the NFD was higher (in an attempt to meet the mEq value of 250) than the requirement (0.2%), we observed higher water consumption relative to when the Na+ concentration was 0.2%. The major issue is that it is implausible to formulate an NFD that meets Na+, K+, and Cl− requirements as well as meeting the mEq value of 250. One of the pertinent questions is which of these 2 is more important. Based on our observation, meeting the requirements for Na+, K+, and Cl− should be given preference over meeting the mEq of 250. 3. Environmental and bacterial influence (gut health) on EAA flow. Most of the studies conducted to determine EAA were carried out in battery cages, which is a cleaner environment compared with litter, where the possibility of exposure to high levels of pathogens exists. The question is whether EAA losses values from birds raised in cages results in an underestimation of losses from birds raised in litter. 4. Region of ileum for sampling. The section of the ileum from where digesta are collected may be important, especially because variations exist between laboratories (entire ileum or the lower half

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5.

6. 7.

8.

Adedokun et al.

of the ileum). One of the downsides of feeding NFD is the low digesta quantity, which makes collection from the entire ileum the more reasonable of the 2 options. However, our recommendation is that standardization should be based on digesta collected from a similar section (e.g., entire ileum vs. distal ileum) of the ileum until it has been empirically shown otherwise. Level of intake (for semipurified diets; e.g., NFD or HDP diets). Dry matter intake may be one of the major factors driving the variations observed in EAA losses. Studies are needed to determine the effect of different levels of DM intake on estimated EAA losses. Effect of relative proportion of energy source (corn starch, dextrose, sucrose, and so on) on EAA flow. Extent of differences between ingredient-specific EAA vs. basal EAA losses. The need exists for comprehensive data on the difference in EAA values estimated using either the NFD or the regression method for different feed ingredients. The difference in EAA values between both methods may indicate the contribution of dietspecific EAA flow to values from the regression method. Additionally, the question remains as to whether diet-specific EAA losses would be additive in a complete ration. Digestive marker. Issues surrounding the use of different markers could be addressed in a single study and based on the result, a case could be made for the choice of a particular marker.

IMPLICATIONS AND FUTURE DIRECTIONS The ability of poultry nutritionists to reduce N excretion, improve flock uniformity, and improve net profit is based on the ability to formulate diets that closely meet the nutrient requirements of birds. This can be accomplished by reducing the safety margins that have been built into diet formulation on a TAA basis. This can be achieved by formulating diets on digestible amino acid basis. Diet formulation on digestible amino acid basis would allow nutritionists to consider amino acid digestibility values of different feed ingredients when formulating diets. In arriving at this, the standardized ileal amino acid digestibility values of various feed ingredients that are sources of amino acids have to be determined, which relies on age-appropriate EAA flow data. Information on standardized ileal amino acid digestibility of individual ingredients will minimize variability that is often associated with feed ingredients as a result of different factors, including processing techniques (as seen in meat and bone meal, soybean meal, and so on) and season or location of the crop. Ileal EAA values for birds at different ages or production phases that are generated from the same species or strains of birds will be required. Additionally, based on the review of existing literature and our work,

the NFD method seems to be the most consistent and reliable method for estimating EAA flow. Finally, the importance of EAA on diet formulation on digestible amino acids is enormous. Consistency in EAA loss data can be greatly increased if some of the issues raised in this review can be addressed and harmonized.

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