Basal ileal endogenous amino acid flow in broiler chickens as influenced by age M. Barua,* M.R. Abdollahi,* F. Zaefarian,* T. J. Wester,* C. K. Girish,y P. V. Chrystal,z and V. Ravindran*,1 *Monogastric Research Centre, School of Agriculture and Environment, Massey University, Palmerston North 4442, New Zealand; yNutrition and Care, Animal Nutrition, Evonik (SEA) Pte. Ltd, 609927 Singapore; and zBaiada Poultry Pty Limited, Pendle Hill NSW 2145, Australia ABSTRACT The current study was carried out to measure the basal ileal endogenous amino acid (EAA) flow in male broilers (Ross 308) at different ages (d 7, 14, 21, 28, 35, and 42), following the feeding of a nitro- gen-free diet. Titanium dioxide (5 g/kg) was included as an indigestible marker. The nitrogen-free diet was offered for four days prior to ileal digesta collection to 6 replicate cages housing 14 (d 3−7), 12 (d 10−14), 10 (d 17−21), 8 (d 24−28), 8 (d 31−35), and 6 (d 38−42) birds per cage. The basal EAA flow was calculated as g/ kg DM intake. The amino acid (AA) profile of endoge- nous protein, expressed as g/100 g protein, was also cal- culated. The basal endogenous flow of nitrogen and all individual and total AA decreased quadratically (P < 0.05 to 0.001), with flows being higher on d 7, then � 2021 The Authors. Published by Elsevier Inc. on behalf of Poultry Science Association Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/). Received February 16, 2021. Accepted May 13, 2021. 1Corresponding author: V.Ravindran@massey.ac.nz 1 decreasing on d 14, plateauing until d 35 and decreasing further on d 42. The concentrations of Trp, Cys, and Gly in the endogenous protein increased linearly (P < 0.01 to 0.001) with advancing age, whereas a linear decrease (P < 0.001) was noted for Lys. A quadratic influence (P < 0.05 to 0.001) was observed for the con- centrations of Ile, Leu, Met, Val, and Asp. These changes in the endogenous protein profile may be attrib- uted to variations in the contribution of endogenous sources with age but delineating the exact contribution of different sources is complicated. Overall, the current findings suggest that the basal ileal EAA flow is influ- enced by broiler age and age-specific EAA flows may need to be considered to standardize the AA digestibility. Key words: age, endogenous flow, amino acids, broilers 2021 Poultry Science 100:101269 https://doi.org/10.1016/j.psj.2021.101269 INTRODUCTION Continuous flow of significant quantities of endoge- nous protein occurs during the process of digestion of ingested feed in poultry (Ravindran, 2021). The sources of endogenous protein are various digestive secretions (bile, gastric, pancreatic, and intestinal secretions), mucoproteins and desquamated epithelial cells lining the gastrointestinal tract (GIT). Accurate quantifica- tion of the flow of endogenous amino acids (EAA) is necessary to calculate the standardized amino acid (AA) digestibility of feed ingredients. These inevitable flows also contribute to the metabolic costs associated with protein synthesis and turnover in the GIT, and to the determination of protein and AA requirements by the factorial method. The measurement and a better understanding of the factors influencing the EAA flow is relevant in improving protein or AA utilization, and thereby reducing nitrogen (N) excretion into the envi- ronment (Cowieson et al., 2009). Endogenous AA losses are influenced primarily by dry matter intake (DMI) and secondarily by the composi- tion of the feed ingredient or diet. These 2 fractions are categorized as basal and specific EAA losses, respec- tively (Ravindran, 2016). Basal endogenous losses are defined as those closely associated with the metabolic functions of the animal and are independent of the diet type. These losses represent the minimum losses that can be expected under any feeding situation. Several methods are available to determine the basal EAA flows in poultry including the regression method, feeding a N- free diet (NFD) or a diet containing highly digestible protein, the peptide alimentation (enzyme hydrolyzed casein) method, or the use of fasted birds. Each of these approaches has its advantages and limitations (Ravindran and Bryden, 1999; Bloke et al., 2017; Par- sons, 2020). Of these, basal ileal EAA estimates deter- mined following the feeding of an NFD is now accepted as being valid for the correction of apparent AA https://doi.org/10.1016/j.psj.2021.101269 http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ mailto:V.Ravindran@massey.ac.nz Table 1. Composition of the nitrogen-free diet (g/kg, as fed basis). Ingredients g/kg Corn starch 842 Cellulose1 50 Soybean oil 50 Dicalcium phosphate 19 Limestone 13 Dipotassium phosphate 12 Titanium dioxide2 5.0 Trace mineral premix3 3.0 Vitamin premix3 2.0 Sodium bicarbonate 2.0 Sodium chloride 2.0 1Ceolus, Microcrystalline Cellulose, Asahi Kasei Corporation, Tokyo, Japan. 2Merck KGaA, Darmstadt, Germany. 3Provided per kilogram of diet: antioxidant, 100 mg; biotin, 0.2 mg; cal- cium pantothenate, 12.8 mg; cholecalciferol, 0.06 mg; cyanocobalamin, 0.017 mg; folic acid, 5.2 mg; menadione, 4 mg; niacin, 35 mg; pyridoxine, 10 mg; transretinol, 3.33 mg; riboflavin, 12 mg; thiamine, 3.0 mg; dl- a-tocopheryl acetate, 60 mg; choline chloride, 638 mg; Co, 0.3 mg; Cu, 3.0 mg; Fe, 25 mg; I, 1 mg; Mn, 125 mg; Mo, 0.5 mg; Se, 0.2 mg; Zn, 60 mg. Table 2. Composition and calculated analysis (g/kg, as fed basis) of broiler starter and finisher diets. Ingredients Starter diet (0−21 d) Finisher diet (22−42 d) Corn 574.2 660 Soybean meal, 46% 381.4 295.6 2 BARUA ET AL. digestibility values (Stein et al., 2007; Ravindran et al., 2017; Parsons, 2020). The NFD is generally composed of starch or sugar (>80%), fortified with insoluble fiber (e.g., cellulose), minerals and vitamins. The assumption is that, since no protein is fed, all N and AA in the ileal digesta are of endogenous origin and represent the basal flows. Feeding an NFD is a simple method despite suffering from limita- tions of underestimation and nonphysiological feeding (Donkoh and Moughan, 1999; Stein et al., 2007). A wide range of factors including the class of birds (broilers, layers, roosters; Ravindran and Hen- driks, 2004), protein status, BW, health status, and DMI are reported to influence the basal EAA estimates (Lemme et al., 2004; Adedokun et al., 2007a). Possible age effect may be an important factor. However, no pre- vious studies have compared the EAA flows throughout the growth cycle of broilers. To the author’s knowledge, except those by Adedokun et al. (2007a,b), all previous studies (Ravindran et al., 2004; Golian et al., 2008; Soleimani et al., 2010; Kong and Adeola, 2013) have the measured EAA flow at a single age using broilers older than 21 d. The applicability of EAA data derived from one single age to all broiler ages is questionable and, therefore, the aim of present study was to determine the basal ileal endogenous N and AA flows at different ages of broilers. Soybean oil 8.8 13.6 Limestone 11.3 9.9 Dicalcium phosphate 10.7 8.2 DL-methionine 3.3 3.0 L-lysine HCl 2.0 1.9 L-threonine 1.0 0.7 Sodium bicarbonate 2.7 2.5 Sodium chloride 2.5 2.5 Trace mineral premix1 1.0 1.0 Vitamin premix1 1.0 1.0 Phytase 0.1 0.1 MATERIALS AND METHODS The experimental procedures were in accordance with the New Zealand Revised Code of Ethical Conduct for the use of live animals for research, testing, and teach- ing, approved by Massey University Animal Ethics Committee. Calculated analysis AME (kcal/kg) 2900 3030 CP 225 190 Digestible lysine 11.0 9.2 Digestible methionine 6.2 5.6 Digestible methionine + cysteine 9.2 8.3 Digestible threonine 7.2 6.0 Crude fat 32 39 Diets The composition of the NFD is shown in Table 1. Titanium dioxide was included as an indigestible marker. Crude fibre 29.3 27.5 Calcium 9.8 8.5 Available phosphorus 4.9 4.2 Sodium 2.2 2.1 Chloride 2.3 2.3 Potassium 11.5 9.7 1Provided per kilogram of diet: antioxidant, 100 mg; biotin, 0.2 mg; cal- cium pantothenate, 12.8 mg; cholecalciferol, 0.06 mg; cyanocobalamin, 0.017 mg; folic acid, 5.2 mg; menadione, 4 mg; niacin, 35 mg; pyridoxine, 10 mg; transretinol, 3.33 mg; riboflavin, 12 mg; thiamine, 3.0 mg; dl- a-tocopheryl acetate, 60 mg; choline chloride, 638 mg; Co, 0.3 mg; Cu, 3.0 mg; Fe, 25 mg; I, 1 mg; Mn, 125 mg; Mo, 0.5 mg; Se, 0.2 mg; Zn, 60 mg. Birds and Housing A total of 500, day-old male broilers (Ross 308) were obtained from a commercial hatchery, raised in floor pens and fed a commercial crumbled broiler starter diet (AME, 2,900 kcal/kg; CP, 225 g/kg) from d 1 to 21 and a broiler finisher diet (AME, 3,030 kcal/kg; CP, 190 g/ kg) in pelleted form from d 22 to 42 (Table 2). Of the 500, 348 healthy birds were used for the experiment. The starter, finisher and NFD diets, in mash form, were offered ad libitum and fresh drinking water was avail- able at all times. During the first week, the average tem- perature was 32 § 1°C and gradually reduced to 23°C by the end of the third week. There were 6 groups of different ages (d 3−7, 10−14, 17−21, 24−28, 31−35, and 38−42) of broilers. On d 3, 84 birds were weighted individually and allocated to 6 battery brooders (n = 14 chicks per replicate) in such a way that the average bird weight per cage was similar. The remaining chicks (n = 264) were raised in floor pens until they were weighed and allocated to 6 replicate grower cages per age group on d 10 (n = 12 birds per cage), d 17 (n = 10 birds per cage), d 24 (n = 8 birds per cage), d 31 (n = 8 birds per cage), and d 38 (n = 6 birds AGE INFLUENCE ON ENDODENOUS AMINO ACID FLOW 3 per cage), respectively. In each age group, the NFD was fed for 4 d (d 3−7, 10−14, 17−21, 24−28, 31−35, and 38−42) prior to the ileal digesta collection. The floor pens, battery brooder and grower cages were housed in an environmentally controlled room with 20 h of fluorescent illumination per day. Determination of Ileal Nutrient Digestibility The birds were euthanized by intravenus injection (1 mL per 2 kg body weight) of sodium pentobarbitone solution (Provet NZ Pty. Ltd., Auckland, New Zealand) on d 7, 14, 21, 28, 35, and 42. Digesta were collected from the lower half of the ileum, as described by Ravindran et al. (2005). The ileum was considered as the portion of the small intestine from Meckel’s diverticulum to a point about ~40 mm proximal to the ileocecal junc- tion. The ileal digesta were collected from all birds into plastic containers by gentle flushing by distilled water, pooled within a cage, immediately frozen, and subse- quently freeze dried (Model 0610, Cuddon Engineering, Blenheim, New Zealand). The diet and freeze-died digesta samples were ground to pass through a 0.5-mm sieve. The samples were stored in airtight plastic contain- ers at 4°C until the analysis of DM, titanium (Ti), N, and AA. Chemical Analysis The DM was determined using the standard proce- dure (Method 930.15; AOAC International, 2016). Tita- nium was measured on an ultraviolet spectrophotometer following the method of Short et al. (1996). Nitrogen was analyzed by combustion (Method 968.06; AOAC International, 2016) using a CNS-200 carbon, N, sulfur analyzer (LECO Corporation, St. Joseph, MI). Amino acids were analyzed following the standard procedures (Method 994.12; AOAC International, 2011). In brief, the samples were hydrolyzed with 6 N HCl (con- taining phenol) for 24 h at 110 § 2°C in glass tubes in an oven. The AA was measured using AA analyzer (ion exchange) with ninhydrin postcolumn derivatization. The chromatograms were integrated using dedicated software (Agilent Open Lab software, Waldbronn, Baden-W€urttemberg, Germany) with AA simulta- neously detected at 570 and 440 nm. Cysteine and methionine were determined as cysteic acid and methio- nine sulphone, respectively, by oxidation with performic acid for 16 h at 0°C and neutralization with hydrobromic acid prior to hydrolysis. For Trp analysis, the samples were saponified under alkaline conditions with barium hydroxide solution in the absence of air at 110°C for 20 h in an autoclave. Fol- lowing alkaline hydrolysis, the internal standard a-methyl Trp was added to the mixture. After adjusting the hydrolysate to pH 3.0 and diluting with 30% metha- nol, Trp and the internal standard were separated by reverse phase chromatography on a HPLC column. Detection was selectively done by means of a fluores- cence detector to prevent interference by other AA and constituents. Calculations All data were expressed on a DM basis for calcula- tions. The basal EAA flow at the terminal ileum was cal- culated as g AA flow per kg DMI using the following formula (Moughan et al., 1992). Basal EAA flow g=kgDMIð Þ ¼ AAconcentration in ileal digesta g=kgð Þ � DietTi g=kgð Þ=Ileal digestaTi g=kgð Þ½ � The AA profile of endogenous protein was calculated by expressing each AA as g/100 g of endogenous protein (N £ 6.25), as indicated below. AA composition of endogenous protein ¼ Endogenous AA=Endogenous CPð Þ � 100 Data Analysis Data were analyzed by the GLM procedure of SAS (version 9.4; 2015; SAS Institute, Cary, NC). Orthogo- nal polynomial contrasts were performed to determine the linear and quadratic effects of broiler age. Cage served as the experimental unit. Statistical significance was declared at P < 0.05. RESULTS No evidence of intestinal abnormalities was observed when the abdominal cavity was opened following eutha- nasia. Feed Intake Feed intake (FI) increased (quadratic, P < 0.001) with advancing age of birds. The average daily FI of birds were 11.4 (d 7), 27.3 (d 14), 49.1 (d 21), 79.6 (d 28), 80.9 (d 35), 79.1 (d 42) g/bird, respectively. Basal Endogenous Flow of Nitrogen and Amino Acids The basal ileal endogenous flow of N and AA at differ- ent ages (d 7, 14, 21, 28, 35, and 42) of broiler, expressed as g/kg DMI, is presented in Table 3. A quadratic influence (P < 0.05 to 0.001) was observed on the basal endogenous flow of N and all AA. The flows were higher on d 7, then decreased on d 14 and plateaued until d 35. After d 35, a further decrease to d 42 was observed. The highest and lowest values were recorded on d 7 and 42, respectively (Figures 1 and 2). Table 3. Basal ileal endogenous nitrogen (N) and amino acid flows1 (g/kg DM intake) at different ages of broilers. Age (days) Orthogonal polynomial contrasts Parameter 7 14 21 28 35 42 Pooled SEM Linear Quadratic N 3.599 1.866 1.793 1.823 1.808 1.295 0.1943 0.001 0.001 Indispensable amino acids Arg 0.678 0.303 0.313 0.358 0.320 0.203 0.0411 0.001 0.007 His 0.294 0.160 0.148 0.160 0.150 0.100 0.0176 0.001 0.012 Ile 0.626 0.325 0.323 0.343 0.301 0.201 0.0352 0.001 0.013 Leu 0.974 0.485 0.490 0.545 0.485 0.314 0.0574 0.001 0.017 Lys 0.644 0.301 0.283 0.321 0.280 0.175 0.0388 0.001 0.004 Met 0.266 0.123 0.124 0.140 0.118 0.069 0.0157 0.001 0.013 Thr 1.352 0.733 0.713 0.667 0.712 0.523 0.0751 0.001 0.002 Trp 0.206 0.116 0.121 0.127 0.121 0.088 0.0129 0.001 0.048 Val 0.811 0.431 0.433 0.453 0.420 0.299 0.0458 0.001 0.009 IAA 5.852 2.975 2.948 3.114 2.906 1.973 0.3365 0.001 0.007 Dispensable amino acids Ala 0.745 0.366 0.361 0.408 0.370 0.248 0.0437 0.001 0.008 Asp 1.407 0.733 0.721 0.744 0.721 0.497 0.0785 0.001 0.005 Cys2 0.474 0.270 0.257 0.249 0.263 0.206 0.0251 0.001 0.001 Glu 1.707 0.799 0.804 0.890 0.809 0.531 0.0985 0.001 0.004 Gly2 0.776 0.397 0.399 0.425 0.399 0.284 0.0437 0.001 0.005 Pro 0.911 0.479 0.481 0.476 0.473 0.341 0.0509 0.001 0.004 Ser 1.061 0.554 0.559 0.536 0.563 0.401 0.0579 0.001 0.002 DAA 7.081 3.599 3.583 3.729 3.559 2.508 0.3966 0.001 0.004 TAA 12.93 6.574 6.531 6.842 6.505 4.481 0.7328 0.001 0.005 1Each value represents the mean of six replicates (14, 12, and 10 birds per replicate for 7, 14, and 21-d old birds, respectively; eight birds per replicate for 28, 35-d old birds; and six birds per replicate for 42-d old birds). 2Semi-indispensable amino acids for poultry.Abbreviations: DAA, total endogenous flow of dispensable amino acids; IAA, total endogenous flow of indispensable amino acids; TAA, total endogenous flow of all amino acids. Figure 1. Basal ileal endogenous amino acid flow of selected indis- pensable amino acids (bars represent means § standard error) as influ- enced by broiler age (Quadratic effects, P < 0.05 to 0.01). Figure 2. Basal ileal endogenous amino acid flow of selected dis- pensable amino acids (bars represent means § standard error) as influ- enced by broiler age (Quadratic effects, P < 0.05 to 0.01). 4 BARUA ET AL. Ileal Digesta Concentrations of Nitrogen and Amino Acids The N and AA concentrations in the ileal digesta of birds fed the NFD, expressed as g/100g digesta, are sum- marized in Table 4. The concentrations of N and all AA, except Trp and Cys, linearly decreased (P < 0.05 to 0.001) as the broilers grew older. Amino Acid Profile of Endogenous Protein The AA profile of ileal endogenous protein, expressed as g/100 g protein, in broilers of different ages is shown in Table 5. The most abundant AA in the endogenous protein was Glu, Asp, Thr, Ser, Leu, Pro, Val, and Ala. The lowest concentration among all AA was recorded for Trp, followed by Met. The concentration of Trp, Cys and Gly linearly (P < 0.01 to 0.001) increased with advancing age, whereas that for Lys linearly (P < 0.001) decreased. Quadratic age effects (P < 0.05 to 0.001) were observed for the con- centration of Ile, Leu, Met, Val, and Asp, but the responses at different ages were variable and inconsis- tent. No age effect was observed for the concentrations of other AA. DISCUSSION The aim of the current study was to investigate whether the basal ileal endogenous flows of N and AA are influenced by the age of broilers. The results showed Table 4. Amino acid concentrations1 (g/100 g of digesta) in the ileal digesta of broilers fed NFD at different ages. Age (days) Orthogonal polynomial contrasts Parameter 7 14 21 28 35 42 Pooled SEM Linear Quadratic N 1.072 0.884 0.957 1.001 0.848 0.725 0.0663 0.003 0.344 Indispensable amino acids Arg 0.202 0.145 0.168 0.197 0.149 0.114 0.0157 0.005 0.234 His 0.088 0.076 0.079 0.088 0.069 0.056 0.0072 0.009 0.152 Ile 0.187 0.155 0.174 0.189 0.141 0.113 0.0144 0.003 0.066 Leu 0.289 0.233 0.263 0.299 0.227 0.176 0.0227 0.007 0.075 Lys 0.192 0.143 0.152 0.176 0.129 0.098 0.0146 0.001 0.326 Met 0.079 0.058 0.067 0.077 0.055 0.039 0.0059 0.001 0.078 Thr 0.404 0.349 0.381 0.366 0.335 0.294 0.0294 0.019 0.487 Trp 0.061 0.055 0.066 0.069 0.057 0.049 0.0055 0.279 0.059 Val 0.242 0.205 0.233 0.249 0.198 0.167 0.0183 0.019 0.100 IAA 1.743 1.418 1.583 1.711 1.359 1.105 0.1313 0.006 0.165 Dispensable amino acids Ala 0.222 0.174 0.194 0.224 0.172 0.139 0.0159 0.007 0.154 Asp 0.419 0.349 0.386 0.409 0.338 0.278 0.0293 0.007 0.166 Cys2 0.141 0.128 0.137 0.137 0.124 0.115 0.0091 0.074 0.444 Glu 0.508 0.381 0.431 0.489 0.377 0.297 0.0368 0.003 0.229 Gly2 0.231 0.189 0.214 0.233 0.188 0.159 0.0166 0.018 0.160 Pro 0.271 0.229 0.258 0.261 0.223 0.191 0.0204 0.021 0.255 Ser 0.317 0.264 0.299 0.295 0.264 0.225 0.0221 0.018 0.338 DAA 2.109 1.714 1.919 2.049 1.688 1.404 1.4881 0.009 0.220 TAA 3.853 3.132 3.502 3.760 3.047 2.509 0.2799 0.008 0.193 1Each value represents the mean of six replicates (14, 12, and 10 birds per replicate for 7, 14, and 21-d old birds, respectively; eight birds per replicate for 28, 35-d old birds; and six birds per replicate for 42-d old birds). 2Semi-indispensable amino acids for poultry.Abbreviations: DAA, total endogenous flow of dispensable amino acids; IAA, total endogenous flow of indispensable amino acids; TAA, total endogenous flow of all amino acids. Table 5. Amino acid composition of endogenous protein1 (g per 100 g crude protein) at different ages of broilers. Age (days) Orthogonal polynomial contrasts Parameter 7 14 21 28 35 42 Pooled SEM Linear Quadratic Indispensable amino acids Arg 3.031 2.439 2.679 2.992 2.883 2.551 0.1069 0.415 0.922 His 1.304 1.359 1.243 1.367 1.348 1.232 0.0422 0.462 0.253 Ile 2.780 2.658 2.763 2.942 2.712 2.479 0.0401 0.004 0.001 Leu 4.335 3.971 4.178 4.622 4.367 3.910 0.1028 0.582 0.031 Lys 2.869 2.429 2.366 2.695 2.532 2.217 0.1139 0.018 0.832 Met 1.189 0.985 1.045 1.200 1.078 0.859 0.0357 0.001 0.031 Thr 5.952 6.284 6.234 5.874 6.287 6.353 0.1724 0.274 0.747 Trp 0.923 0.967 1.039 1.096 1.074 1.086 0.0343 0.001 0.115 Val 3.599 3.583 3.751 3.927 3.764 3.683 0.0569 0.035 0.008 Dispensable amino acids Ala 3.312 3.028 3.124 3.569 3.319 3.106 0.0803 0.676 0.189 Asp 6.244 6.093 6.331 6.489 6.428 6.169 0.0861 0.297 0.049 Cys2 2.092 2.345 2.314 2.192 2.310 2.540 0.0572 0.001 0.379 Glu 7.616 6.504 6.889 7.547 7.279 6.613 0.1668 0.171 0.808 Gly2 3.443 3.269 3.497 3.697 3.571 3.527 0.0455 0.002 0.091 Pro 4.043 4.068 4.227 4.111 4.212 4.179 0.1046 0.273 0.599 Ser 4.684 4.689 4.919 4.699 4.916 4.909 0.1097 0.109 0.914 1Each value represents the mean of six replicates (14, 12, and 10 birds per replicate for 7, 14, and 21-d old birds, respectively; eight birds per replicate for 28, 35-d old birds; and six birds per replicate for 42-d old birds). 2Semi-indispensable amino acids for poultry.Abbreviations: IAA, total endogenous flow of indispensable amino acids; DAA, total endogenous flow of dispensable amino acids; AAA, total endogenous flow of all amino acids. AGE INFLUENCE ON ENDODENOUS AMINO ACID FLOW 5 that the basal ileal endogenous N and AA flows were markedly higher on d 7 and, then declined on d 14 and plateaued until d 35. A further decrease was observed on d 42. Compared to d 7, the basal endogenous N flow declined by 48 and 64% on d 14 and d 42, respectively. The endogenous flow of all AA on d 7 was almost twice of the values from d 14 to 35, and three times higher than the value on d 42. In agreement with the current findings, following the feeding of a NFD to broilers, Adedokun et al. (2007a) recorded approximately 2 times higher ileal EAA flow on d 5 (8.69 g/kg DMI) compared to d 15 (3.73 g/kg DMI) and 21 (3.95 g/kg DMI), with no significant differences between the flows on d 15 and 21. Possible explanations for the higher ileal EAA flows determined on d 7 and reduced flows with advancing age are intricate. A proportion of secreted endogenous pro- teins is digested, along with ingested dietary proteins, in the GIT and absorbed. The EAA values determined at the ileum therefore relate to the algebraic differences between that secreted and absorbed (Moughan, 2003; Ravindran et al., 2004). The source of endogenous pro- teins and the entry point into the GIT further complicate 6 BARUA ET AL. this dynamic (Ravindran, 2021). When digestive enzymes dominate the endogenous flow, the proteins pass through the duodenum and jejunum where there is greater oppor- tunity for digestion and absorption. In contrast, if mucus secretion or desquamation is significant, particularly if they occur distal to the duodenum, then the opportunity for digestion is lower and there will be relatively higher endogenous losses at the ileal level. Owing to this compli- cated dynamic, it is difficult to differentiate the true con- tribution of each endogenous source. Nevertheless, the ramification is that the higher EAA losses on d 7 may reflect increased secretion, reduced absorption, or both. The first week after hatch is the most critical period in the life of a broiler chicken. At hatching, the digestive system of the chick is still immature and there is the transition from yolk to oral nutrition. Substan- tial physical and functional development of the GIT and digestive organs take place during the first week. It is therefore clear that the capacity to digest the feed and, absorb and transport nutrients is limited during this period. Thus, reduced absorption, rather than increased secretion, is the likely reason for the greater ileal EAA losses determined at d 7. Against this background, several possibilities may be considered as discussed below. First, the GIT of the newly hatched chick is immature and the bird places high priority on intestinal growth to ensure the development of nutrient supply functions, as evidenced by dramatic growth during the first week (Sklan, 2001). In the days following hatching, weights of proventriculus, gizzard, and small intestine increase more rapidly in relation to BW than other organs and tissues (Katanbaf et al., 1988; Sell et al., 1991). This enhanced growth is maximal in chicks between 4 and 8 d of age and thereafter there is a relative decline. The mass of the small intestine increases almost 600% within the first 7 d (Noy et al., 2001). The length of the small intestine and its individual component regions also increase with age. According to Iji et al. (2001), the rela- tive weight of small intestine peaked between d 7 and 14. The rapid growth in intestinal size during the first week may lead to mechanical inefficiency in the passage and mixing of digesta, reducing nutrient digestion, and increasing the EAA flow. Second, the function of the GIT is strictly related to its microscopic structure. The architecture of GIT is not well developed during the first week of life but rapidly develops with age. The dramatic post-hatch increases observed in the weight and length of small intestine is reported to be minor relative to the growth of gut mucosa (Dibner et al., 1996). Uni et al. (1995) found that the villus height and area in the chick increase rap- idly at different rates in different intestinal segments, particularly in the jejunum and ileum from 4 to 10 d post-hatch. Crypt depth, which reflects enterocyte mat- uration rate, increased linearly in both the duodenum and jejunum until 10 d. A study of morphological devel- opment of GIT in broiler chicks by Uni et al. (1996) showed that the height and perimeter of villi increased by 34 to 100% in all small intestinal segments between 4 and 10 d after hatching. In addition, the crypt depth, the enterocytes number per longitudinal section of villi increased with increasing age. The poorly developed intestinal architecture will also lead to lower digestion during week 1. Third, the secretion of digestive enzymes by pancreas and the brush border of the small intestine is low at the time of hatch (Sell, 1996), but increase after hatch although the rate of increase was different for different enzymes (Tarvid, 1995; Noy and Sklan, 1997). The rela- tive activity of aminopeptidases in all intestinal segments reduces after hatching, reaches a low point on d 10 and increase on d 15 (Tarvid, 1992). The greatest activity of dipeptidase was found at hatching and decrease within 7 days by 25% (Tarvid, 1990). The specific activity of pan- creatic trypsin is lower during the first 3 to 6 d post-hatch, increases afterwards by up to 20% on d 14 (Nitsan et al., 1991). Both the relative trypsin and chymotrypsin activi- ties (unit per kg BW) in pancreas increases with age, reaching a maximum level on d 11. The activity of trypsin in small intestine contents increases 10-fold from hatching to d 14. In case of chymotrypsin, it increases 3-fold to a maximum at 20 d of age, suggesting that lower protease activities during the first week after hatch could limit the AA digestibility. However, after wk 1, the trypsin activity increases up to 21 d, with no noticeable changes afterward (Nitsan et al., 1991). In summary, during wk 1, the GIT is in a maturation phase in terms of growth, morphology and, secreted amounts and activities of proteolytic enzymes. Taken together, these imply a compromised protein digestion increasing the endogenous AA recovery at the ileal level. Some other relevant factors potentially contributing to the higher EAA flow at d 7 may be worth considering. Cell proliferation and cell turnover are greater during wk 1 (Iji et al., 2001) and desquamated cells may there- fore be important contributors to the EAA flow at d 7. There is some evidence that, when an NFD is fed, the source of EAA is largely mucoproteins (Adedokun et al., 2007a). The intestinal mucous layer is a protective bar- rier against harmful intraluminal components and microflora (Gork et al., 1999). Mucin plays a significant role in filtering nutrients in GIT and thus affecting the absorption of nutrients (Smirnov et al., 2006). Mucin glycoproteins are rich in Thr, Ser, Pro, Glu, and Asp (Souffrant, 1991; Lien et al., 1997). Adedokun et al. (2007b) reported higher Thr and Ser contents in the ileal digesta of 5-day-old broiler chickens compared to d 15 and 21 following feeding an NFD, reflecting a higher concentration of intestinal mucin at early ages. Similarly, in the present study, higher mucin production in the newly hatched chick was manifested by the higher endogenous flows of Thr (102%), Asp (106%), Glu (123%), Pro (103%), and Ser (103%) on d 7 than the average of other ages (d 14−42). Intestinal cells and mucin are poorly digested, and this may explain the higher EAA recovery at d 7. Lower relative mucin secre- tion and, increased endogenous protein digestion and absorption with age (Nasset, 1972; Ravindran and Bry- den, 1999) may account for the lower endogenous N and AA secretion in older birds. AGE INFLUENCE ON ENDODENOUS AMINO ACID FLOW 7 Food transit time through the digestive tract is an important factor influencing the nutrient digestion by determining the available time for contact among nutrients, digestive enzymes, absorptive surfaces, and microbiome (Clemens et al., 1975; Mateos et al., 1982; Vergara et al., 1989). Longer digesta retention increases the absorption of nutrients by enhancing contact time with absorptive cells (Washburn, 1991). Several studies have recorded slower intestinal passage rate in adult birds (Hurwitz and Bar, 1966; Sklan et al., 1975; Noy and Sklan, 1995). Noy and Sklan (1995) reported a longer digesta passage time in broiler chicks on d 4 than on d 14 (160 vs. 110 min). Decreased passage rate with age allows the digesta to be exposed to the digestive and absorptive processes for a longer time that may improve nutrient digestibility, including AA, in older birds. Since all N in the ileal digesta in birds fed the NFD diet come only from endogenous proteins, one may argue that the AA concentration in the ileal digesta, expressed as g/kg and not corrected for DMI, may give some repre- sentation of the dynamic in EAA flow with age. When DMI was excluded, the concentration of almost all AA in the ileal digesta decreased with advancing broiler age. Since the AA originated from endogenous sources, this observation may suggest increased re-absorption of endogenous protein with age. In present study, FI increased gradually from 11.4 g/bird on d 7 to 79.1 g/bird on d 42, which might account, to some extent, for the observed differences in basal EAA flows, expressed as g/kg DMI, among broiler ages. It must be noted, however, the primary aim of measuring EAA losses is to standardize the apparent digestibility values and this correction will not be possible when EAA values are not linked to the DMI. The concentrations of EAA in the ileal digesta are within the range reported in the literature (Ravindran, 2021). The higher proportions of Glu, Asp, Thr, and Ser determined in the endogenous protein were as expected. Asp, Leu, Ser, andGlu are themajorAA in the ileal endogenous secretions in chickens (Bielorai and Iosif, 1987; Siriwan et al., 1994; Adedokun et al. 2007b). Ravindran et al. (2004) reported that themajorAA in endogenous protein in the ileal digesta of 35-day-old chickens fed an NFD was Thr, Asp, and Glu. Tarvener et al. (1981) speculated that the high proportions of Glu, Asp, Thr, Ser, and Leu in the endogenous protein may be due to their slower rate of re-absorption compared with other AA. The lower concentrations of Met and His can be attributed to the fact that these AA are absorbed in greatest proportions compared to others in the GIT (Webb, 1990). It is generally assumed that the AA profile of endoge- nous protein is somewhat constant, although it is known that individual sources of endogenous protein have dif- ferent AA composition (Ravindran, 2021). The current findings suggest that the composition of the AA flow was altered by broiler age, ostensibly reflecting changes in the contribution of endogenous protein components with age. The concentration of Cys, Gly, and Trp was linearly increased with age. Cys is found in relatively higher concentrations in the mucin (Lien et al., 1997). The Gly was previously assumed to be originating from biliary secretions (Ravindran and Bryden, 1999; Ravindran and Hendriks, 2004; Adedokun et al., 2011), but current evidence indicates that the bile acid conju- gation in poultry is exclusively with taurine and not with Gly as in pigs (Ravindran, 2021). The inconsistent effects of age on Ile, Leu, Met, Val, and Asp are difficult to explain and, as mentioned earlier, underline the com- plexity of identifying the exact contribution of different endogenous components. CONCLUSIONS The current findings, for the first time, provide informa- tion on the basal endogenous AA flows in broilers from hatching to 42 d of age. The flow was higher on d 7, reduced on d 14 and remained constant until d 35. A fur- ther reduction was noticed on d 42. The higher EAA flow at d 7 is ostensibly reflective of the immature digestive sys- tem and the resultant low digestive capacity in the newly hatched broiler chick. Factors like longer digesta reten- tion, improved AA digestibility and, relatively lower pro- duction and increased utilization of mucin may account for the lower basal EAA flows in older birds. Therefore, the use of a single EAA value for birds of all ages will underestimate the standardized ileal AA digestibility in early life and overestimate the digestibility in older birds. Application of age-specific values for EAA flowmay bene- fit accurate standardization of amino acid digestibility and improve the precision of feed formulations. ACKNOWLEDGMENTS The authors wish to express their appreciation to the “AgriFutures Australian Chicken Meat Program” for funding the project, and to Preethi Ramesh, AMINO- Lab EMSEA, Singapore for AA analyses. DISCLOSURES Authors declare no conflict of interest. REFERENCES Adedokun, S. A., O. Adeola, C. M. Parsons, M. S. Lilburn, and T. J. Applegate. 2011. 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http://refhub.elsevier.com/S0032-5791(21)00303-5/sbref0054 http://refhub.elsevier.com/S0032-5791(21)00303-5/sbref0054 Basal ileal endogenous amino acid flow in broiler chickens as influenced by age INTRODUCTION MATERIALS AND METHODS Diets Birds and Housing Determination of Ileal Nutrient Digestibility Chemical Analysis Calculations Data Analysis RESULTS Feed Intake Basal Endogenous Flow of Nitrogen and Amino Acids Ileal Digesta Concentrations of Nitrogen and Amino Acids Amino Acid Profile of Endogenous Protein DISCUSSION CONCLUSIONS ACKNOWLEDGMENTS DISCLOSURES REFERENCES