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Effect of Age and Method on Ileal Endogenous Amino Acid Flow in Turkey Poults¹

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

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

² Corresponding author: applegt{at}purdue.edu

	ABSTRACT

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Ileal endogenous amino acid (IEAA) flow in turkey poults was determined at 2 experimental locations on d 5, 15, and 21 posthatch using 3 methods, namely a N-free diet (NFD), a highly digestible protein (casein), and the regression method, obtained by regressing IEAA flow against dietary casein levels. The diets were semipurified and contained 0, 50, 100, or 150 g of casein/kg of diet as the sole source of dietary protein. Each diet was fed for 5 d to 6 replicate cages of 30 (d 5), 10 (d 15), or 8 (d 21) birds per cage. There was no interaction between locations and age or locations and diet, so the data from both locations were pooled. Ileal endogenous amino acid flow on d 5 (NFD method) was higher (P < 0.05) than on d 15 or 21. Ileal endogenous amino acid flow estimated from the NFD and the regression methods was different on d 5 (P < 0.05), but there were no differences in IEAA flow for most of the amino acids on d 15 and 21. Increasing the level of casein resulted in a linear (P < 0.05) increase in IEAA flow. The amino acids with the lowest flow were Trp and Met, whereas Glu had the greatest flow. The results obtained from this study indicate that data generated across laboratories were repeatable. The results also suggest that about twice as much amino acids of endogenous origin are found in the digesta of poults on d 5 relative to d 21. Also, as poults age, there is a decrease in IEAA flow from d 5 to 15, but flows from d 15 to 21 are not different. These observations suggest that apparent digestibility coefficients for poults on d 5 and d 15 or 21 could be significantly influenced by the level of endogenous amino acids, more so on d 5.

Key Words: casein • endogenous amino acid flow • nitrogen-free diet • regression • turkey poult

	INTRODUCTION

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The need to optimize amino acid utilization in poultry by formulating diets on a digestible amino acid basis is becoming much more important with the increased focus on decreasing nutrient excretion, and this has been discussed in several recent publications (De Lange et al., 2003; Lemme et al., 2004). The advantages include more closely meeting the actual requirements of the bird while taking into consideration the digestibility of amino acids from various feed ingredients. This can only be achieved, however, if amino acids of dietary and endogenous origins are distinguished.

Many studies have been conducted in chicks and adult chickens of different ages and strains using a variety of methods to estimate amino acids of endogenous origin (Siriwan et al., 1993; Ravindran et al., 2004; Ravindran and Hendriks, 2004). From these studies, the different methods of estimating ileal endogenous amino acid (IEAA) flow resulted in different estimates of endogenous amino acid concentrations. Endogenous amino acid flows from fasted cockerels have been reported to be lower than the flow from birds fed a N-free diet (NFD; Muztar and Slinger, 1980). Likewise, the presence in the gut of increasing levels of dietary amino acids of dietary origin have been reported to have resulted in increasing endogenous amino acid secretion in broilers and cockerels (Siriwan et al., 1993). The origin of endogenous amino acids varies and may include amino acids from the digestive enzymes of salivary, gastric, and biliary secretion; mucoproteins; and sloughed cells (Ravindran and Hendriks, 2004).

Given that an apparent digestibility coefficient includes both the basal as well as the diet-induced flow of an endogenous amino acid, a factor is needed to delineate the contribution of amino acids of endogenous origin; hence, the determination of IEAA becomes important. There are no published data for IEAA flow in poults of any age. The objective of the current study, therefore, was to examine the effects of age, location, and method on basal IEAA flow in turkey poults. These basal endogenous flows can be used to determine standardized ileal amino acid digestibility coefficients for different feed ingredients.

	MATERIALS AND METHODS

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Diet Formulation
Four semipurified diets were formulated to contain graded concentrations of casein (0, 50, 100, or 150 g/ kg of diet). The dietary ingredient composition of the experimental diets and calculated values for some nutrients are reported in Table 1. For the IEAA and total amino acid (TAA) flow calculations, chromic oxide was added to the treatment diets as an indigestible marker at 3 g/ kg of diet.

Birds were raised in battery cages (Alternative Design Manufacturing and Supply Inc., Siloam Springs, AR) and maintained in an environmentally controlled room with 24 h of light throughout the duration of the study. The room temperature was 35°C during the first week and was reduced to 25°C by wk 3. Birds had free access to feed and water. All animal care procedures were approved by the Purdue University and The Ohio State University Animal Care and Use Committee.

Sampling and Ileal Digesta Processing
On d 5, 15, and 21, the contents from the ileal region between Meckel’s diverticulum to about 5 mm proximal to the ileo-cecal junction region were flushed with distilled water. For birds sampled on d 5, a 50-mL syringe was used, whereas a wash bottle was used for flushing on d 15 and 21. The ileal digesta samples from all the poults within a cage were pooled, frozen, and stored at –20°C until they were processed. All frozen samples were freeze-dried and ground using mortar and pestle.

Chemical Analysis
The DM content was determined on the ground diets and ileal digesta by drying the samples at 100°C for 24 h. Amino acid and Cr analyses were conducted at the University of Missouri Experiment Station and Chemical Laboratory. For amino acid analyses, samples were hydrolyzed in 6 N HCl for 24 h at 110°C under N atmosphere. For the S-containing amino acids, Met and Cys, performic acid oxidation was carried out before acid hydrolysis. For Trp analysis, samples were hydrolyzed using barium hydroxide. The amino acids in the hydrolyzate were 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 and perchloric acid wet ash digestion.

Calculations
Ileal endogenous amino acid flow and TAA (sum of all amino acids and taurine) was calculated as milligrams of amino acid flow per kilogram of DM feed intake using the formula proposed by Moughan et al. (1992a):

Statistical Analysis
The data were analyzed using the GLM procedure of SAS (SAS Inst. Inc., Cary, NC). Orthogonal polynomial contrasts were used to compare the treatment means for the effects of dietary casein concentration, whereas relevant treatment means with significant F-ratios were separated using Tukey adjustment. Ileal endogenous amino acid flow determined by the regression method was obtained by regressing IEAA or TAA flow against dietary casein concentration. A comparison of the IEAA and TAA flows between the NFD and regression methods was made by calculating the standard errors of difference of means as outlined in Samuels and Witmer (1999). The probabilities were determined using the t-test.

	RESULTS

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The analyzed dietary amino acids are reported in Table 2. The level of total dietary amino acids increased from 1.2 to 138.7 g/kg of diet from 0 (NFD) and 150 g of casein/ kg of diet, respectively (Table 2). There was generally no significant interaction between experimental location and age and location and diet; hence, data from both locations were pooled. The interaction between location and dietary treatments was not tested on d 15, because data (for d 15) from 1 of the 2 locations were discarded from the entire analysis because of errors in ileal digesta collection method (squeezing as against flushing with distilled water). For diets containing graded levels of casein, it was assumed that all protein was digested and absorbed. The mean values for IEAA flow when the NFD was fed as well as estimates from the regression method are presented in Table 3. The IEAA flow on d 5 was higher (P < 0.05) than on d 15 or 21. The TAA and IEAA flow for most of the amino acids on d 15 or d 21 was less than 40% of the flow on d 5. Methionine and His were the amino acids with the lowest values, whereas the flows of Glu and Asp were the highest. The extrapolated IEAA and TAA flows to zero percent casein in the diet on d 15 and 21 were less than 50% of that on d 5 (Table 3). However, IEAA flow on d 15 and 21 was similar. The ileal endogenous flow of all the amino acids on d 5 was different, with the NFD method resulting in higher (P < 0.05) IEAA flow relative to values from the regression method. On d 15, however, only 3 amino acids (Met, Thr, and Cys) were different (P < 0.05). Theonine, Val, Cys, Gly, and Pro were the only amino acids whose flows were higher (P < 0.05) with the NFD method relative to the regression method on d 21.

	DISCUSSION

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The influence of age on IEAA flow is obvious from the results of these studies. Irrespective of the method used, the flow of amino acids of endogenous origin was higher on d 5 than on d 15 or 21. The values on d 15 and 21, however, were similar. The very high output of IEAA on d 5 could be attributed to intestinal secretions of digestive enzymes, sloughed epithelial cells lining the gastrointestinal tract (Moughan et al., 1992b) as well as a rapid rate of intestinal cell proliferation and turnover within the first week of age (Uni, 1999). The fact that the IEAA flow was higher on d 5 or slightly lower on d 15 and 21 when the NFD method was compared with the 50 g of casein/ kg diet could be an indication that the presence of protein in the diet increased IEAA flow on d 15 and 21 while decreasing flow on d 5 (data not shown). It is also evident from the results of this study that increasing the level of dietary casein increased the level of IEAA output. This observation is supported by the findings of Brannon (1990) and Siriwan et al. (1993), who suggested that increasing levels of dietary amino acids could increase the endogenous secretion of digestive enzymes as well as increase sloughing of cells lining the intestinal wall. To examine the validity of the assumption of complete digestibility and absorption of amino acids and peptides from dietary casein, we standardized apparent amino acid digestibility at the 3 ages evaluated in this study using the IEAA flow from the birds fed the NFD. This resulted in standardized digestibility values of greater than 94% for most of the amino acids irrespective of the levels of dietary casein. Also, there was no linear effect of casein level on standardized ileal digestibility, although there was linear effect for the apparent digestibility values (data not shown). The linear increase in IEAA flow with increase in dietary casein observed in this study may be as a result of increased intestinal secretions arising from increased dietary protein in the gut. Mariscal-Landin and Reis de Souza (2006) fed graded levels of casein (from 55.3 to 221.3 g/kg of diet) to weaned and growing pigs and observed apparent casein digestibility to be greater than 90% at the highest level of casein (221.3 g/kg of diet) inclusion for most of the amino acids.

The interaction between location and age was also determined for each of the dietary treatments. It can be inferred from the results of this study that the lack of interaction between location and age, except for Thr (for the 50 g of casein/kg diet), supports the conclusion that the methods of determining IEAA flow used in this study are repeatable across experimental locations. The lack of interaction between location and age is strongest for the NFD method, which indirectly suggests that it could be the method that is most likely to be repeatable and consistent across laboratories.

When amino acids of endogenous origin were determined by extrapolating a regression line to zero percent protein intake, values obtained for d 5 were about 30% less than the values obtained using the NFD method. On d 15 and 21, IEAA flows from the regression method were not significantly different than those of the NFD for most of the amino acids. The results from the comparison of the 2 methods (NFD and the regression) suggest that at the younger age (d 5), the 2 methods will give different results, with the NFD method resulting in a higher flow estimate. At older ages, however, the 2 methods resulted in similar endogenous amino acid flow with the exception of a few amino acids. This observation is different from what Siriwan et al. (1993) reported in 5-wk-old broilers. They observed a significantly higher IEAA flow when the regression method was used relative to the NFD method.

Increasing concentration of casein in the diets from 0 to 150 g/kg of diet resulted in a linear increase in IEAA flow. When the IEAA from the 21-d-old poults used in this study was compared with the IEAA flow in 5-wk-old broilers (Ravindran et al., 2004), a similar trend was observed, especially when using the NFD method. However, when the 150 g of casein/kg diet in the current study was compared with the diets containing 190 g of enzyme-hydrolyzed casein and 194 g of guanidinated casein (Ravindran et al., 2004), the IEAA flow in the chicks was higher than what was observed for poults in this study. Many factors may have contributed to this including age differences, species differences, and differences in the levels of dietary CP. Hence, it is probable that not all protein in the diet was digested and absorbed or an increasing level of dietary protein elicited an increase in the level of endogenous amino acid secretion in the gut. Most of the individual IEAA flow values determined using the NFD methods were similar in both studies. The level of ileal TAA flow in 21-d-old turkey poults fed the 150 g of casein/kg diet in this study (12,078 mg/kg of DM intake) is similar to what was reported by Ravindran and Hendriks (2004) for 6-wk-old broiler chicks using the peptide alimentation method (12,305 mg/kg of DM intake). Likewise, there was no interaction between location and dietary treatments, which underscores the fact that in birds of similar age, fed the same diet and using similar analytical procedures, the effect of experimental location is not significant.

Glutamic acid, Asp, Leu, Thr, Val, Pro, and Ser were the amino acids with the highest endogenous flow, independent of the method used. These amino acids (except Leu) have been reported to be high in mucin, and because little digestion of mucin takes place before the distal end of the gastrointestinal tract (Lien et al., 1997), it would be expected that their concentrations in the digesta may be high relative to the other amino acids. An increase in these amino acids, especially Thr, with an increasing concentration of dietary casein may be an indication of an increase in the level of mucin production with increasing dietary amino acids. This could be due to the need to protect the intestinal lining from the increasing levels of digestive enzymes being secreted in response to increased level of dietary proteins in the gut.

In summary, our results clearly show that age has a significant effect on IEAA flow, with d-5 flows being about twice those of d 15 or 21. It is interesting to note, however, that in this study, IEAA flow on d 21 compared favorably with previously published IEAA flow in 5-and 6-wk-old broilers. This may be an indication that IEAA flow based on DMI from wk 2 to 6 may not be very different. This also suggests that correcting for endogenous amino acid flow at early ages (<10 d) may result in raising amino acid digestibility coefficients relative to when standardization is done on d 21. Ileal endogenous amino acid flow is also method-dependent, with an increase in IEAA flow concomitant with increasing concentrations of 100 and 150 g of casein in the diets. The regression and NFD methods resulted in flow estimates of different magnitudes on d 5 with higher flows from the NFD method, but there were no differences on d 15 and 21 for most amino acids. Higher IEAA flows on d 5 could explain in part the low apparent N digestibility obtained in turkey poults and broiler chicks during the first week. Formulating diets based on standardized ileal digestibility coefficients of amino acids will consider the high flow of amino acids of endogenous origin, which may eventually lead to a decrease in N excretion, especially within the first week. The results from this study also show that low apparent amino acid digestibility coefficients of feed ingredients during the first week may be attributed to the high level of endogenous amino acid flows.

	ACKNOWLEDGMENTS

 
Partial funding for this project was supplied by the 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 2007-18075 of the Purdue University Agricultural Research Programs.

Received for publication January 18, 2007. Accepted for publication April 29, 2007.

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