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Comparison of Ileal Endogenous Amino Acid Flows in Broiler Chicks and Turkey Poults¹

S. A. Adedokun^*, C. M. Parsons, M. S. Lilburn, O. Adeola^* and T. J. Applegate^*,2

^* Department of Animal Sciences, Purdue University, West Lafayette, IN 47907; Department of Animal Sciences, University of Illinois, Urbana-Champaign 61801; and Department of Animal Sciences, The Ohio State University, Ohio Agricultural Research and Development Center, Wooster 44691

² Corresponding author: applegt{at}purdue.edu

	ABSTRACT

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Ileal endogenous amino acid (IEAA) and total amino acid (TAA) flow (mg/kg of DM intake) in turkey poults and broiler chicks at 3 ages (5, 15, and 21 d) were compared by feeding a N-free diet (NFD) or graded levels of casein (highly digestible protein, HDP). The semipurified diets contained 0 (NFD), 50, 100, or 150 g of casein/kg of diet as the only source of amino acids. Each diet was fed to 6 replicate cages containing 30 (5 d), 10 (15 d), or 8 (21 d) birds per cage for 5 d prior to the collection of ileal digesta. At d 5, IEAA and TAA flow in poults fed the NFD, 50, and 100 g of casein/kg of diet was higher (P < 0.05) than in chicks. The IEAA flows within and between both species on d 15 and 21 were not different. Similar trends were observed for the HDP diets (50, 100, or 150 g of casein/kg of diet). An interaction (P < 0.05) between species and age was observed for most of the amino acids for all treatments. The results from this study suggest that at d 5, poults have significantly higher concentration of IEAA and TAA output relative to chicks. However, by d 15 and 21, there were no species differences in IEAA or TAA flow. These results also showed that IEAA flow is species and age dependent. The increased IEAA flow observed at d 5 should be taken into consideration when formulating starter diets on a digestible amino acid basis.

Key Words: broiler chick • casein • ileal endogenous amino acid • nitrogen-free diet • turkey poult

	INTRODUCTION

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The goal of poultry nutrition is to optimize animal performance by feeding diets that will meet the bird’s requirements while minimizing dietary cost and nutrient excretion. Lemme et al. (2004) highlighted the importance of standardizing amino acid digestibility coefficients of feed ingredients. To accurately formulate diets using digestible amino acid coefficients, it is important to have an estimate of those amino acids that are of endogenous origin. These values can subsequently be used to standardize apparent digestibility coefficients for a wide range of feed ingredients.

Several methods have been used to estimate the concentration of amino acids of endogenous origin. These estimates have been derived by ad libitum feeding of the N-free diet (NFD; Siriwan et al., 1994; Ravindran et al., 2004), precision feeding using adult cecectomized roosters (Parsons, 1986; Siriwan et al., 1993), and the use of the regression method (Siriwan et al., 1993, 1994). Other methods include the peptide alimentation (enzyme hydrolyzed casein) method (Darragh et al., 1990; Butts et al., 1993; Ravindran and Hendriks, 2004) and homoarginine method using guanidination reaction (Siriwan et al., 1994), feeding of low level of highly digestible protein (HDP, casein; Siriwan et al., 1994; Ravindran et al., 2004), as well as the use of isotope markers (de Lange et al., 1990). However, each of the aforementioned methods has some limitations. A review of endogenous amino acid production in growing pigs (Nyachoti et al., 1997) and in poultry (Ravindran and Bryden, 1999) listed the strengths and weakness of each of these methods, but any of them can be used to standardize digestibility estimates by serving as a correction factor. In addition to this, Kluth et al. (2005) and Kluth and Rodehutscord (2006) have shown that the linear regression method can be used to standardize digestibility coefficient without the need for separate quantification of endogenous losses. This, according to Rutherford et al. (2004), will remove the intra- and interlaboratory variations that are associated with endogenous loss estimation.

The original source of ileal endogenous amino acids (IEAA) includes protein from desquamated epithelial cells lining the gastrointestinal tract, serum albumin, mucoprotein, and various digestive secretions (Moughan et al., 1992a; Nyachoti et al., 1997). The method of ileal digesta collection (squeezing, flushing, etc.) also affects the amount of amino acid of endogenous origin found in ileal digesta. The effects of age and strain of birds (broilers, layers, and adult roosters) on IEAA flow have been reported (Ravindran and Hendriks, 2004, Ravindran et al., 2004). However, there are few data on IEAA flow in broiler chicks and turkey poults at very early ages (the first 3 wk), nor are there any data that compare IEAA or TAA flow in these species.

The objective of this study was to determine and compare the IEAA and TAA flow in broiler chicks and turkey poults at 3 ages (d 5, 15, and 21) using 2 different methods of IEAA and TAA determination. The methods used in this study incorporated NFD and HDP diet. The hypothesis tested was that the IEAA flow is dependent on species, age, and method of determination.

	MATERIALS AND METHODS

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Diet Formulation
Four semipurified diets were used in this study. The diets included NFD (0 g of casein/kg of diet) and diets containing 50, 100, or 150 g of casein (International Ingredient Corporation, St. Louis, MO)/kg of diet (Table 1). Casein was the sole source of protein (amino acids) in the diets. Two diets (conventional broiler starter and turkey starter diets) were also made and fed to the respective species prior to the treatment diets. The starter diets for the 2 species were primarily corn-soy diets and were formulated to meet or exceed the NRC (1994) recommendations. Chromic oxide (3 g/kg of diet) was added to the treatment diets and used as an indigestible marker. The same diet batch was used for the 2 studies. The analyzed dietary amino acid and TAA contents (on DM basis) are reported in Table 2.

Each experimental diet was fed for 5 consecutive days before ileal contents were collected. Six replicate cages containing 30, 10, and 8 birds per cage were euthanized by carbon dioxide asphyxiation and the ileal contents collected by flushing with distilled water on d 5, 15, and 21, respectively. The ileum was considered to be the region between Meckel’s diverticulum and 1 cm proximal to the ileocecal junction. Birds that were sampled on d 15 and 21 were fed the respective starter diets until d 10 and 16 when 240 and 192 birds from each species were randomly assigned to individual cages.

Birds were raised in battery cages (Alternative Design Manufacturing and Supply Inc., Siloam Springs, AR) in an environmentally controlled room with 24 h of light. The rearing temperature was 35°C during the first week, and the temperature was reduced by 5 degrees during each subsequent week. Birds had ad libitum access to feed and water, and all animal care procedures were approved by the Purdue University Animal Care and Use Committee.

Sampling and Ileal Digesta Processing
On d 5, 15, and 21, the digesta within the ileum was removed by flushing with distilled water. For those birds sampled on d 5, a 50-mL syringe was used to flush, whereas a wash bottle was used on d 15 and 21. The ileal digesta samples from all the birds within a cage were pooled and frozen (–20°C) until they were processed. Samples were freeze-dried, ground with a mortar and pestle, and analyzed for complete amino acid profile and chromium.

Chemical Analysis
Dry matter content was determined on all dried and ground diets and ileal digesta by drying the samples at 100°C for 24 h. Amino acids and chromium analyses were conducted at the University of Missouri Experiment Station Chemical Laboratories. Prior to amino acid analyses, all samples were hydrolyzed in 6 N HCl for 24 h at 110°C under N atmosphere. For Met and Cys, performic acid oxidation was carried out before acid hydrolysis. The samples used for tryptophan analysis were hydrolyzed using barium hydroxide. The amino acids in the hydrolysate were then determined by HPLC after postcolumn derivatization (AOAC, 2000; method 982.30 E [a, b, c]). Amino acid concentrations were not corrected for incomplete recovery resulting from hydrolysis. Chromium was determined by the inductively coupled plasma atomic emission spectroscopy method (AOAC, 2000; method 990.08) following nitric/perchloric acids wet ash digestion.

Calculations
Ileal endogenous amino acid and TAA flow from both species was calculated as milligrams of amino acid or TAA flow per 1 kg of DM intake using the following formula by Moughan et al. (1992b):

Statistical Analysis
Data were analyzed using the PROC GLM procedure of SAS (SAS Inst. Inc., Cary, NC) with species and age as the class variables. Where F-ratios indicate significance, treatment means were separated using Tukey adjustment. Level of significance was set at P < 0.05.

	RESULTS

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Mean BW in grams at sampling (NFD) was chick, 47, poult, 57 (d 5); chick, 180, poult, 168 (d 15); chick, 312, poult, 318 (d 21). When 50 g of casein/kg of diet was fed, mean BW in grams at sampling (chick vs. poult) at d 5, 15, and 21 were 53 vs. 61, 192 vs. 167, and 318 vs. 327, respectively. Mean BW in grams at sampling when 100 g of casein/kg of diet was fed (chick vs. poult) were 58 vs. 64 (d 5), 203 vs. 180 (d 15), and 343 vs. 343 (d 21). The mean BW in grams for birds on 150 g of casein/kg of diet at sampling were 63 vs. 65 (d 5), 225 vs. 198 (d 15), and 363 vs. 362 (d 21) for chick and poult, respectively.

The IEAA flow from the chicks and poults fed the NFD is reported in Table 3. At d 5, the IEAA flow was higher (P < 0.05) in poults than in chicks. The percentage TAA flow in chicks on d 5, 15, and 21 were 46, 66, and 60%, respectively, of the TAA flow observed for poults at the same ages. Glutamic acid, Asp, and Leu were the amino acids with the greatest flow in both species on d 5. On d 15 and 21, Glu, Asp, and Thr had the greatest flow. Methionine and Thr flow in chicks on d 5, 15, and 21 were, respectively, 39 and 48%, 65 and 70%, and 52 and 59% of endogenous Met and Thr flow in poults at the same age. The flow for TAA was not significantly different between the 2 species on d 15 and 21. Similar trends were seen in all the amino acids as well. The interaction between age and species was significant for all the amino acids when the NFD method was used.

	DISCUSSION

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According to Mitchell (1924), endogenous N represents the N found in digesta, feces, or both, when animals are fed a NFD. A number of studies have been conducted to determine the IEAA and N flow in chicks and adult chickens (Ravindran and Hendriks, 2004; Ravindran et al., 2004). Basal EAA loss can also be determined from the intercept estimate of regression equation (Kluth and Rodehutscord, 2006). However, to the authors’ knowledge, no studies have been conducted with very young (5-d) broiler chicks, and there are no data on IEAA flow in turkey poults and no published comparison of IEAA flow between the 2 species at any ages. In most of the studies available, IEAA flow determination was conducted with older chickens (i.e., 2-, 4-, or 5-wk-old broiler chickens; Ravindran et al., 2004) and 6-wk-old broilers, 70-wk-old laying hens, and 70-wk-old roosters (Ravindran and Hendriks, 2004). Additionally, method of estimation used included feeding of NFD, guanidinated casein, enzyme hydrolyzed casein (Ravindran et al., 2004), and peptide alimentation (Ravindran and Hendriks, 2004). In this study we determined IEAA and TAA flow at 3 different ages (d 5, 15, and 21) and in 2 poultry species (broiler chicks and turkey poults) using 2 methods (NFD and feeding of HDP methods).

The level of feed intake, especially the level of protein intake, has been reported to be positively correlated with endogenous amino acid flow in growing pigs and rats by increasing endogenous secretion (Darragh et al., 1990; Butts et al., 1993). Also, the presence of protein of dietary origin in the gut may negatively impact the breakdown and reabsorption of endogenous protein (Snook and Meyer, 1964). Body size may also play a role because heavier birds are expected to have higher DM intake coupled with a heavier gastrointestinal tract and increased surface area and IEAA flow due to an accelerated rate of mucin production and sloughing of intestinal cells.

The significantly higher flow in poults compared with chicks on d 5 when NFD was fed as well as a decrease in flow for both species from d 5 to 21 showed species and age differences in IEAA losses. This shows that independent of differences in BW at sampling, poults had higher rates of IEAA and TAA flow (d 5) compared with chicks fed the NFD. Despite the fact that poults were about 10 g (21%) or 6 g (2%) heavier than chicks on d 5 and 21, respectively, the chicks consumed more feed than poults at d 21 (data not shown), but it is difficult to attribute the difference, especially on d 5, to simple differences in BW or feed intake alone.

Increases in the concentration of dietary protein with casein (from 0 to 150 g of casein/kg of diet) resulted in increased IEAA and TAA flow at all 3 ages in both species. In poults, however, the diet containing 50 g of casein resulted in relatively lower IEAA and TAA flow on d 5. The presence of protein in the diet (50 g of casein at age d 5) also resulted in a relative increase in the level of IEAA and TAA flow in chicks with Met, Thr, and TAA flow in chicks being, respectively, about 81, 80, and 89% of that of poults. Unlike observations with the NFD, and 100 g of casein diets, the flow of TAA between the 2 species within any of the 3 ages was not different when the diet contained 50 or 150 g of casein/kg of diet.

The large increases in IEAA flow in chicks when the diets contained graded concentrations of casein suggested that chicks responded to the stimulatory effects of dietary protein by increasing endogenous secretions (digestive enzymes or mucin) or by increasing enterocyte turnover. For instance, when 50 g of casein/kg of diet was fed, the proportion of chick to poult TAA flow increased for d 5, 15, or 21 from 46, 66, or 60% (NFD) to 89, 99, or 89%, respectively. This proportion is similar in almost all the amino acids (Lys, Met, and Thr: NFD, 45, 39, or 48%; 50 g of casein diet, 97, 81, or 80%) on d 5. Glutamic acid flow, although very high, did not differ between chicks and poults on d 5 when 50, 100, or 150 g of casein/kg of diet was fed within each of the 3 ages evaluated. The high proportion of Glu, Asp, Thr, Ser, Leu, and Lys may be seen as a result of slow rate of reabsorption of these amino acids that are of endogenous origin as compared with other amino acids (Taverner et al. 1981). Additionally, Ile, Lys, Pro, Ser, and Tyr flows were not different on d 5 when diets containing 50 or 150 g of casein were fed. The greater increase in IEAA and TAA flow when protein-containing diets were fed could be attributed to an increase in digestive secretion in chicks or a decrease in the rate of digestion and absorption of EAA and TAA relative to poults or vice versa. The decrease in IEAA and TAA flow with age (d 5 vs. d 15 or 21) could be attributed to an improvement in the digestion and reabsorption of amino acids that are of endogenous origin after the first week. The use of EHC in determining IEAA flow when compared with NFD method resulted in higher (2 to 3 times greater) values of IEAA (Ravindran et al., 2004).

Free amino acids and small peptides have been reported to be readily absorbed in pigs leaving the bulk of IEAA and TAA flow to be composed of mucin proteins due to their resistance to enzymatic hydrolysis (Moughan and Schuttert, 1991). Based on this report, the high IEAA flow in 5-d-old turkey poults may have been due to a higher amount of mucin (as evidenced by Thr and Ser endogenous flow) production relative to chicks, which was evident whether IEAA flow determination was by NFD or HDP. When fed NFD, the level of Lys in the digesta of chicks was about half of the poults; however, this difference was greatly reduced when HDP was fed, especially the diet containing 50 g of casein.

From the review of the development of small intestine in domestic birds, Uni (1999) reported that the intestine morphological development in chicks was faster than in poults. The fact that the rate of enterocyte development in poults only catches up with that of chicks after the 12th day (Uni, 1999) is an indication that the high level of endogenous amino acid flow in poults on d 5 relative to chicks must have been greatly influenced by factors other than the sloughing of intestinal epithelium or the rate of cell turnover. The significantly high levels of Thr, Ala, Asp, Gly, Pro, and Ser in poults on d 5 could be attributed to a higher level of mucin production. In addition to the fact that chicks responded to the presence of protein in the diet by increasing the secretion of amino acids of endogenous origin (compared with poults), the lack of a difference in IEAA flow between the 2 species at d 15 and 21 (for all levels of casein) could be explained more by a larger decrease in EAA flow in poults by d 15 and 21 (relative to d 5) than a higher increase in EAA flow in chicks as a result of increased dietary protein.

In a study conducted in our lab to determine the standardized ileal amino acid digestibility coefficients of some plant feed ingredients (corn, soybean meal, canola meal, and 2 types of distillers dried grains with solubles) using NFD method, the increase in digestibility was higher for poults than for chicks and is an indication that poults had higher basal endogenous amino acid flow than chicks, especially on d 5. However, these improvements vary with ingredients. For example, the standardized ileal amino acid digestibility coefficients for TAA (soybean meal) increased by 4.3% (d 5) and 1.33% (d 21) in chicks, whereas the improvement in poults was 12% (d 5) and 8% (d 21, unpublished data).

The results from this study show that IEAA and TAA flow is age, species, and method dependent. More IEAA and TAA were produced at younger age (d 5) in both species relative to d 15 and 21. Turkey poults produced greater level of IEAA and TAA relative to broiler chicks on d 5; however, the difference between the 2 species disappeared between d 15 and 21. Also, the relative level of IEAA and TAA flow is a function of the method used. Increasing level of endogenous amino acid flow was observed with increasing level of dietary casein. Finally, correction for endogenous amino acid secretion is needed especially at younger ages, and the effect of dietary protein on IEAA and TAA flow is more pronounced in young chicks than poults.

	ACKNOWLEDGMENTS

 
Partial funding for this project was supplied by US Poultry and Egg Association, Fats and Proteins Research Foundation, Degussa Corporation, ADM Inc., Novus International Inc., and Ajinomoto Heartland, LLC.

	FOOTNOTES

 
¹ Journal paper number 2006-17946 of the Purdue University Agricultural Research Programs.

Received for publication December 2, 2006. Accepted for publication April 16, 2007.

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