Open Access Anim Biosci Vol. 34, No. 10:1632-1642 October 2021 https://doi.org/10.5713/ab.20.0838 pISSN 2765-0189 eISSN 2765-0235 Addition of hydrochloric acid to collection bags or collection containers did not change basal endogenous losses or ileal digestibility of amino acid in corn, soybean meal, or wheat middlings fed to growing pigs Su A Lee1, Laia Blavi1,2, Diego M. D. L. Navarro1,3, and Hans H. Stein1,* * Corresponding Author: Hans H. Stein Objective: The hypothesis was that apparent ileal digestibility (AID), basal endogenous Tel: +1-217-333-0013, E-mail: hstein@illinois.edu losses, and standardized ileal digestibility (SID) of amino acids (AA) are not affected by adding acid to collection containers or bags used to collect ileal digesta from pigs. 1 Department of Animal Sciences, University of Methods: Twenty-four growing barrows (initial body weight: 77.8±4.5 kg) that were fitted Illinois, Urbana, IL 61801, USA 2 Departament de Ciència Animal i dels with a T-cannula in the distal ileum were fed diets for three 7-d periods. An N-free diet and Aliments, Universitat Autònoma de 3 diets containing corn, soybean meal, or wheat middlings as the sole source of AA were Barcelona, Barcelona, Spain used. Within each period, each of the 4 diets were fed to 6 pigs. Among the 6 pigs, digesta 3 Hamlet Protein Inc., Findlay, OH 45840, USA from 3 pigs were collected in bags containing no HCl, whereas 40 mL of 3 N HCl was ORCID included in the bags used to collect digesta from the remaining 3 pigs. Every other bag Su A Lee collected from each pig was emptied into a container without adding HCl, whereas the https://orcid.org/0000-0001-9351-7196 remaining bags were added to a container along with 40 mL of 3 N HCl for each bag. All Laia Blavi https://orcid.org/0000-0003-4292-7245 digesta were stored at –20°C immediately after collection. Data were analyzed using a model Diego M. D. L. Navarro that included feed ingredient, HCl in bags, HCl in containers, and all 2-way and 3-way inter­ https://orcid.org/0000-0002-8680-3845 actions as fixed effects. No 3-way interactions were significant, and data were, therefore, Hans H. Stein https://orcid.org/0000-0002-4855-661X reanalyzed independently for each diet as a 2×2 factorial. Results: There were no interactions between adding HCl to collection bags and to containers, Submitted Dec 15, 2020; Revised Jan 27, 2021; and no effects of adding HCl to collection bags or containers for AID, basal endogenous Accepted Feb 26, 2021 losses, or SID of most AA were observed. Conclusion: It is not necessary to add acid to digesta collection bags or collection containers if ileal digesta are stored at –20°C immediately after collection. Keywords: Amino Acids; Collection; Digestibility; Hydrogen Chloride; Ileal Digesta; Pigs INTRODUCTION Dietary protein is digested in the small intestine of pigs and free amino acids (AA) or small peptides are absorbed before the end of the small intestine. Therefore, ileal digestibility is the most accurate estimation of AA digestibility because by measuring ileal digestibility interference by microbes in the hind-gut is avoided [1]. However, there is also microbial fermentation in the small intestine of pigs [2], and ileal digesta, therefore, contain micro- bial protein [3]. Therefore, AA in ileal digesta may be metabolized unless microbial action is stopped immediately after collection. In most AA digestibility experiments collection bags are changed at least every 30 min and the collected ileal digesta are stored at –20°C to prevent microbial degradation of N. Another attempt to stop microbial action in the digesta includes adding acids to collection bags [4,5] or to collection containers [6,7] to reduce pH of ileal digesta. To our knowledge, Copyright © 2021 by Animal Bioscience This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, 1632 and reproduction in any medium, provided the original work is properly cited. www.animbiosci.org Lee et al (2021) Anim Biosci 34:1632-1642 there is, however, no information about the need for adding this experiment. Pigs were allotted to 4 diets with 6 pigs being acids to collected digesta and it is not known if values for ap- fed each of the 4 diets in each of the 3 periods. Pigs were parent ileal digestibility (AID), basal endogenous losses of housed in individual pens (1.2×1.5 m) and each pen was AA, or standardized ileal digestibility (SID) of AA are influ- equipped with a feeder and a nipple drinker and had fully enced by addition of acids to digesta. Therefore, the objective slatted tribar floors and smooth sidewalls. of this experiment was to test the null-hypothesis that values Pigs were fed their assigned diets in a daily amount of 3.4 for AID, basal endogenous losses, and SID of AA by pigs are times the estimated energy requirement for maintenance (i.e., not influenced by the addition of acids to ileal digesta collec- 197 kcal metabolizable energy per kg0.60) [9], and 2 equal tion bags or to collection containers regardless of the ingredient meals were provided every day at 0800 and 1600 h. Water being fed. was available at all times. MATERIALS AND METHODS Treatments A total of 4 diets were formulated (Table 1). Three diets con- The Institutional Animal Care and Use Committee at the tained corn, soybean meal (SBM), or wheat middlings as the University of Illinois reviewed and approved the protocol for only source of protein and AA, and the fourth diet was an the experiment. Pigs used in the experiment were the offspring N-free diet that was used to determine endogenous losses of of L 359 boars mated to Camborough females (Pig Improve- AA (Tables 2, 3). Vitamins and minerals were included in all ment Company, Hendersonville, TN, USA). diets to meet or exceed estimated nutrient requirements for growing pigs [9]. All diets also contained 0.40% chromic oxide Animals, experimental design, housing, and feeding as an indigestible marker. All diets were fed to pigs in mash Twenty-four barrows (initial body weight: 77.8±4.5 kg) that form. were equipped with a T-cannula in the distal ileum [8] were The 24 pigs were allotted to 4 diets and 3 periods using used. Pigs had been used in a different experiment before being fed a common finisher diet for 14 d and then used in Table 2. Analyzed composition of the ingredients, as-is basis Soybean Wheat Table 1. Ingredient composition of experimental diets, as-is basis Item (%) Corn meal middlings Soybean Wheat Dry matter 86.72 89.62 88.89 Ingredient (%) Corn N-free meal middlings Gross energy (kcal/kg) 3,808 4,191 4,053 Ground corn 94.90 - - - Ash 1.01 7.31 4.79 Soybean meal - 38.00 - - Total dietary fiber 9.7 16.0 36.9 Wheat middlings - - 40.00 - Soluble dietary fiber ND 0.8 1.9 Cornstarch - 45.70 42.20 67.22 Insoluble dietary fiber 9.7 15.2 35.0 Soybean oil 2.00 3.60 - 4.00 Acid hydrolyzed ether extract 3.06 1.27 4.07 Ground limestone 0.70 0.65 1.15 0.46 Crude protein 7.52 48.00 16.57 Dicalcium phosphate 1.30 0.95 0.55 1.72 Indispensable amino acids Arg 0.36 3.43 1.09 Sodium chloride 0.40 0.40 0.40 0.40 His 0.22 1.22 0.44 Sucrose - 10.00 15.00 20.00 Ile 0.26 2.13 0.50 Chromic oxide 0.40 0.40 0.40 0.40 Leu 0.87 3.66 1.00 Magnesium oxide - - - 0.10 Lys 0.28 3.00 0.73 Potassium carbonate - - - 0.40 Met 0.15 0.63 0.23 Solka floc1) - - - 5.00 Phe 0.37 2.45 0.65 Vitamin and mineral premix2) 0.30 0.30 0.30 0.30 Thr 0.27 1.82 0.52 1) Fiber Sales and Development Corp., Urbana, OH, USA. Trp 0.04 0.68 0.17 2) The vitamin-micromineral premix provided the following quantities of Val 0.35 2.22 0.74 vitamins and micro minerals per kg of complete diet: vitamin A as retinyl Dispensable amino acids acetate, 11,136 IU; vitamin D3 as cholecalciferol, 2,208 IU; vitamin E as DL-alpha tocopheryl acetate, 66 IU; vitamin K as menadione dimethyl- Ala 0.53 2.03 0.77 primidinol bisulfite, 1.42 mg; thiamin as thiamine mononitrate, 0.24 mg; Asp 0.50 5.17 1.13 riboflavin, 6.59 mg; pyridoxine as pyridoxine hydrochloride, 0.24 mg; Cys 0.16 0.62 0.32 vitamin B12, 0.03 mg; D-pantothenic acid as D-calcium pantothenate, 23.5 Glu 1.34 8.43 2.88 mg; niacin, 44.1 mg; folic acid, 1.59 mg; biotin, 0.44 mg; Cu, 20 mg as Gly 0.30 1.99 0.86 copper sulfate and copper chloride; Fe, 126 mg as ferrous sulfate; I, 1.26 Ser 0.35 1.99 0.59 mg as ethylenediamine dihydriodide; Mn, 60.2 mg as manganese sulfate; Se, 0.3 mg as sodium selenite and selenium yeast; and Zn, 125.1 mg as Tyr 0.21 1.77 0.41 zinc sulfate. ND, not detectable. www.animbiosci.org 1633 Lee et al (2021) Anim Biosci 34:1632-1642 Table 3. Analyzed composition of the experimental diets period was considered an adaptation period to the diets. Ile- Diet al digesta were collected for 8 h (from 0800 to 1600 h) on d 6 Item (%) Soybean Wheat and 7 by attaching a plastic bag to the opened cannula barrel, Corn N-free meal middlings and digesta flowing into the bag were collected and stored in Dry matter 89.09 94.23 94.05 96.19 collection containers at –20°C. Bags were removed whenever Crude protein 6.90 19.96 6.91 0.40 they were filled with digesta or at least once every 30 min. Gross energy (kcal/kg) 3,807 4,003 3,747 3,895 On the completion of one experimental period, animals Indispensable amino acids were deprived of feed overnight and the following morning, Arg 0.33 1.07 0.44 0.01 a new experimental diet was offered. His 0.21 0.39 0.18 ND At the conclusion of the experiment, all containers were Ile 0.25 0.71 0.22 0.01 Leu 0.84 1.22 0.44 0.03 removed from freezers, the ileal digesta were thawed and Lys 0.26 1.00 0.31 0.02 mixed in each container, and a sub-sample was collected for Met 0.13 0.19 0.09 ND chemical analysis. Ileal digesta samples were lyophilized and Phe 0.35 0.81 0.28 0.02 finely ground prior to analysis. Thr 0.26 0.60 0.22 0.01 Trp 0.06 0.29 0.09 < 0.03 Chemical analysis Val 0.33 0.71 0.31 0.01 Based on the methods described in AOAC Int. [11], corn, Dispensable amino acids SBM, wheat middlings, diets, and ileal digesta samples were Ala 0.51 0.68 0.33 0.02 analyzed for dry matter (method 930.15) and ingredients Asp 0.47 1.71 0.47 0.02 Cys 0.15 0.20 0.13 ND were analyzed for ash (method 942.05). Ingredient samples Glu 1.26 2.81 1.28 0.04 were also analyzed for insoluble and soluble dietary fiber using Gly 0.28 0.66 0.36 0.01 the Ankom Dietary Fiber Analyzer (Ankom Technology, Ser 0.32 0.71 0.27 0.01 Macedon, NY, USA; method 991.43). Total dietary fiber was Tyr 0.19 0.52 0.16 0.01 calculated as the sum of soluble and insoluble dietary fiber. ND, not detectable. Acid hydrolyzed ether extract in corn, SBM, and wheat middlings was analyzed by acid hydrolysis using 3 N HCl (AnkomHCl, Ankom Technology, USA) followed by crude the balanced Latin square design program [10]. Thus, 6 pigs fat extraction using petroleum ether (AnkomXT15, Ankom were fed each diet in each period. Among the 6 pigs fed the Technology, USA). Ingredients and diets samples were an- same diet, digesta from 3 pigs were collected in bags con- alyzed for N by combustion (method 999.03) [11] on an taining no HCl whereas 40 mL of 3 N HCl was included in Elementar Rapid N-cube protein/nitrogen apparatus (Ele- the bags used to collect digesta from the remaining 3 pigs. mentar Americas Inc., Mt. Laurel, NJ, USA). Aspartic acid Following collection, digesta bags were alternately emptied was used as a calibration standard and crude protein was into collection containers without adding HCl or into contain- calculated as N×6.25. These samples were also analyzed for ers along with 40 mL of 3 N HCl for each bag. All containers AA on a Hitachi Amino Acid Analyzer (Model L8800, Hit- were stored at –20°C at all times. Therefore, for pigs fed the achi High Technologies America Inc., Pleasanton, CA, USA) 3 diets containing corn, SBM, and wheat middlings, there using ninhydrin for post-column derivatization and nor- were a total of 12 treatments arranged in a 3×2×2 factorial leucine as the internal standard. Samples were hydrolyzed with feed ingredients (i.e., corn, SBM, and wheat middlings) with 6 N HCl for 24 h at 110°C prior to analysis, but Met as the first factor and no HCl or addition of HCl in either and Cys were analyzed as methionine sulfone and cysteic collection bags or collection containers as the second and acid after cold performic acid oxidation overnight before third factors, respectively. For pigs fed the N-free diet, there hydrolysis, and Trp was determined after NaOH hydrolysis were a total of 4 treatments that were arranged in a 2×2 for 22 h at 110°C (method 982.30 E [a, b, c]) [11]. Ingredi- factorial with no HCl or addition of HCl in either collec- ents and diets were analyzed for gross energy using an tion bags or collection containers. Three replicate pigs were isoperibol bomb calorimeter (Model 6400, Parr Instru- used per treatment in each period and there were a total of ments, Moline, IL, USA). Benzoic acid was used as the 9 replications per treatment for the 3 periods. standard for calibration. The chromium concentration in diets and ileal digesta samples was determined using In- Sample collection ductive Coupled Plasma Atomic Emission Spectrometric Pig weights were recorded at the beginning of each period method (method 990.08) [11]. Samples were prepared us- and at the conclusion of the experiment and the amount of ing nitric acid-perchloric acid (method 968.08D[b]) [11]. feed supplied each day was recorded. The initial 5 d of each 1634 www.animbiosci.org Lee et al (2021) Anim Biosci 34:1632-1642 Calculations and statistical analysis therefore, analyzed independently for each ingredient as a Values for the AID, basal endogenous losses, and SID of AA 2×2 factorial using the same split-plot arrangement as used in each diet were calculated as described previously [12]. for the initial model. Data for basal endogenous losses were Four values for basal endogenous losses of AA were deter- also analyzed using this model. Treatment means were cal- mined: from samples stored without or with HCl in collection culated by using the LSMEANS statement and if the interaction bags and without or with HCl in collection containers. Values was significant, the PDIFF option was used to separate means. for SID of AA were calculated by correcting AID values for Statistical significance and tendency were considered at p< each treatment for basal endogenous losses calculated for 0.05 and 0.05≤p<0.10, respectively. the same treatment. Therefore, for corn, SBM, and wheat middlings, there were 4 AID and SID values calculated based RESULTS on the inclusion or not of HCl in collection bags and inclu- sion or not of HCl in collection containers. Pigs remained healthy during the experiment and very little Normality of residuals was verified using the UNIVARIATE feed refusals were observed. procedure (SAS Inst. Inc., Cary, NC, USA). Outliers were identified as values that deviated from the treatment mean Apparent ileal digestibility of amino acids by more than 3 times the interquartile range [13]. Data were For corn, SBM, and wheat middlings, there were no inter- analyzed using a split-plot arrangement in PROC MIXED actions between HCl in collection bags and in collection (SAS Inst. Inc., USA) with the 3 feed ingredients and the 2 containers for the AID of any AA (Tables 4, 5, and 6). No levels of HCl in collection bags as whole-plot factors, and effects of adding HCl to collection bags or collection con- the 2 levels of HCl in collection containers as the split-plot tainers for the AID of AA in corn, SBM, or wheat middlings factor. The initial model included the fixed effects of feed in- were observed with the exception that the AID of Met in gredients, HCl in collection bags, HCl in collection containers, wheat middlings was greater (p = 0.049) if HCl was added all 2-way interactions, and all 3-way interactions. The ran- to collection bags compared with no HCl in the bags. The dom effects were HCl in collection bags and square, period, AID of Met in SBM tended to be greater (p = 0.060) if HCl and pig nested within HCl in collection bags. However, no was added to collection containers than if no HCl was added. 3-way interactions were significant (p>0.10), and data were, The AID of Phe in wheat middlings tended to be greater (p Table 4. Apparent ileal digestibility (%) of amino acids in corn without or with 40 mL of 3 N HCl in the collection bag or the collection container HCl in bag − + p-value Item (%) SEM HCl in container − + − + Bag Container Bag×container Observations (n) 8 8 9 9 - - - - Indispensable amino acids Arg 72.8 73.0 71.0 72.3 6.0 0.875 0.829 0.872 His 79.9 76.6 81.9 81.9 2.5 0.259 0.399 0.384 Ile 73.9 73.5 76.0 77.9 2.2 0.282 0.648 0.463 Leu 84.6 84.1 85.9 86.6 1.4 0.307 0.931 0.565 Lys 70.0 72.8 70.2 72.7 3.3 0.985 0.251 0.951 Met 83.0 83.3 84.8 86.4 1.4 0.179 0.375 0.529 Phe 79.8 79.3 81.5 82.5 1.7 0.242 0.839 0.593 Thr 62.6 61.7 65.1 67.4 3.0 0.305 0.765 0.466 Trp 74.5 74.6 75.3 76.7 2.3 0.616 0.646 0.721 Val 70.7 70.4 73.7 75.1 2.5 0.278 0.724 0.585 Total 76.5 76.2 77.9 79.1 2.1 0.452 0.723 0.568 Dispensable amino acids Ala 75.6 76.5 76.9 78.7 2.0 0.492 0.355 0.771 Asp 69.8 69.7 71.3 72.9 2.4 0.471 0.672 0.623 Cys 71.0 71.1 73.6 74.7 2.3 0.317 0.723 0.752 Glu 81.9 82.0 83.5 84.3 1.5 0.336 0.651 0.750 Gly 24.4 29.4 26.1 25.6 8.8 0.929 0.595 0.523 Ser 73.3 73.0 75.0 75.7 2.0 0.409 0.876 0.742 Tyr 71.3 71.3 74.5 75.1 2.5 0.292 0.862 0.868 Total 56.5 60.6 58.7 57.7 4.9 0.960 0.534 0.326 Total amino acids 66.5 68.4 68.6 68.5 3.3 0.821 0.642 0.608 SEM, standard error of the means. www.animbiosci.org 1635 Lee et al (2021) Anim Biosci 34:1632-1642 Table 5. Apparent ileal digestibility (%) of amino acids in soybean meal without or with 40 mL of 3 N HCl in the collection bag or the collection container HCl in bag − + p-value Item (%) SEM HCl in container − + − + Bag Container Bag×container Observations (n) 9 8 9 9 - - - - Indispensable amino acids Arg 91.7 91.1 89.1 89.8 1.1 0.220 0.990 0.247 His 84.9 86.1 84.6 85.1 1.5 0.742 0.338 0.648 Ile 81.8 82.5 82.8 84.1 1.8 0.618 0.239 0.731 Leu 82.2 82.6 82.8 84.0 1.8 0.703 0.378 0.660 Lys 83.4 84.4 84.7 86.1 1.6 0.515 0.171 0.842 Met 84.4 85.4 84.6 86.5 1.6 0.784 0.060 0.533 Phe 82.4 82.9 83.2 84.3 1.8 0.685 0.345 0.713 Thr 75.6 74.7 75.4 76.5 2.2 0.792 0.934 0.456 Trp 88.8 88.4 88.5 89.1 1.2 0.912 0.856 0.498 Val 77.6 77.5 77.9 79.8 2.5 0.723 0.470 0.459 Total 83.3 83.5 83.3 84.5 1.5 0.807 0.388 0.565 Dispensable amino acids Ala 72.3 73.6 73.9 76.7 3.0 0.573 0.216 0.626 Asp 78.2 79.7 80.4 81.1 1.9 0.499 0.357 0.719 Cys 68.9 69.6 70.4 71.1 3.2 0.736 0.714 0.998 Glu 79.5 81.8 82.1 82.6 2.3 0.586 0.310 0.538 Gly 55.1 57.1 59.6 62.8 4.3 0.375 0.332 0.818 Ser 82.8 80.6 81.0 82.0 1.1 0.872 0.487 0.079 Tyr 83.8 82.9 83.3 84.2 1.8 0.878 0.983 0.320 Total 71.3 73.2 73.1 74.8 2.4 0.589 0.232 0.948 Total amino acids 77.3 78.4 78.3 79.7 1.8 0.620 0.272 0.884 SEM, standard error of the means. Table 6. Apparent ileal digestibility (%) of amino acids in wheat middlings without or with 40 mL of 3 N HCl in the collection bag or the collection container HCl in bag − + p-value Item (%) SEM HCl in container − + − + Bag Container Bag×container Observations (n) 8 9 8 9 - - - - Indispensable amino acids Arg 71.2 72.4 67.3 76.3 6.1 0.999 0.245 0.370 His 70.0 72.4 75.1 76.2 3.2 0.316 0.374 0.746 Ile 62.6 63.3 66.7 66.2 2.7 0.239 0.983 0.815 Leu 67.9 68.3 72.5 71.9 2.2 0.083 0.976 0.826 Lys 68.9 69.5 70.4 70.5 3.0 0.744 0.846 0.905 Met 70.2 69.9 76.2 75.4 2.1 0.049 0.741 0.917 Phe 68.3 68.1 72.5 72.1 2.2 0.085 0.878 0.966 Thr 48.7 48.5 54.4 52.5 4.5 0.293 0.817 0.855 Trp 68.6 69.2 70.9 73.7 3.3 0.377 0.592 0.719 Val 60.6 61.7 65.6 65.1 2.6 0.131 0.912 0.754 Total 65.9 66.3 68.6 69.9 2.8 0.338 0.730 0.872 Dispensable amino acids Ala 44.3 50.4 47.8 55.3 5.0 0.450 0.148 0.885 Asp 60.3 60.5 61.6 63.5 3.3 0.539 0.748 0.784 Cys 64.5 64.9 66.9 68.5 2.9 0.361 0.734 0.838 Glu 79.8 80.3 80.6 82.1 1.5 0.437 0.483 0.727 Gly 3.0 36.3 3.8 25.5 16.1 0.761 0.106 0.723 Ser 61.1 61.0 63.3 64.2 3.7 0.505 0.911 0.883 Tyr 64.7 64.7 67.5 67.0 3.0 0.435 0.931 0.924 Total 27.1 41.9 19.4 42.6 12.8 0.830 0.049 0.644 Total amino acids 45.9 54.3 42.8 56.0 8.0 0.949 0.078 0.680 SEM, standard error of the means. 1636 www.animbiosci.org Lee et al (2021) Anim Biosci 34:1632-1642 = 0.085) if HCl was added to collection bags than if no HCl served among other treatments (interaction; p<0.05). Addition was added. The AID of total dispensable AA in wheat mid- of HCl to collection bags did not change values for SID of dlings was also greater (p<0.05) if HCl was added to collection most AA in SBM and there were no effects of adding HCl to containers than if no HCl was added. collection containers with the exception that the SID of Met was greater (p = 0.041) if HCl was added to collection con- Basal endogenous losses of amino acids tainers than if no HCl was added to containers. For the SID No interactions between the addition of HCl to collection of AA in wheat middlings, no interactions between adding bags and collection containers were observed for basal en- HCl to collection bags and containers were observed (Table dogenous losses of AA (Table 7). There were also no effects 10). Adding HCl to either collection bags or collection con- of adding HCl to either collection bags or collection con- tainers did not affect the SID of AA in wheat middlings. tainers. DISCUSSION Standardized ileal digestibility of amino acids No interactions between adding HCl to collection bags and The direct procedure was used to determine SID of AA in containers for the SID of AA in corn were observed, and corn, SBM, and wheat middlings although this procedure there were no effects of adding HCl to either collection bags results in diets based on corn and wheat middlings having or containers for the SID of AA in corn (Table 8). For SBM, AA concentrations below the requirement for the pigs. How- no interactions between adding HCl to collection bags and ever, feeding diets with AA below the requirement does not containers for the SID of AA except for Arg, Gly, and Ser influence the SID of AA [14]. The three ingredients were were observed (Table 9). Addition of HCl only to collection chosen because they have different concentrations of fiber containers decreased (p<0.05) the SID of Arg and Ser in SBM, and protein and therefore may differently affect ileal digesta but no HCl effect was detected among other treatments (in- characteristics. Concentrations of AA in corn, SBM, and teraction; p<0.05). The SID of Gly was less (p<0.05) if HCl wheat middlings, values for the SID of AA in corn and SBM, was added only to collection containers than if HCl was added and the basal endogenous losses of AA were reasonably close to collection bags and containers, but no difference was ob- to values in the literature [9,15]. The SID of most AA in wheat Table 7. Basal endogenous losses of amino acids (g/kg dry matter intake) without or with 40 mL of 3 N HCl in the collection bag or the collection container HCl in bag − + p-value Item SEM HCl in container − + − + Bag Container Bag×container Observations (n) 9 9 8 9 - - - - Indispensable amino acids Arg 0.60 0.45 0.86 0.86 0.12 0.120 0.290 0.266 His 0.15 0.12 0.13 0.14 0.02 0.965 0.661 0.271 Ile 0.25 0.22 0.17 0.23 0.04 0.440 0.593 0.100 Leu 0.40 0.35 0.28 0.37 0.06 0.516 0.640 0.106 Lys 0.30 0.25 0.18 0.26 0.05 0.411 0.612 0.066 Met 0.06 0.05 0.03 0.05 0.01 0.431 0.778 0.170 Phe 0.26 0.23 0.19 0.24 0.04 0.513 0.644 0.124 Thr 0.47 0.42 0.33 0.44 0.07 0.518 0.453 0.103 Trp 0.08 0.07 0.07 0.08 0.01 0.880 0.749 0.140 Val 0.35 0.31 0.24 0.32 0.05 0.508 0.588 0.149 Total 2.92 2.48 2.41 2.99 0.36 0.994 0.783 0.062 Dispensable amino acids Ala 0.60 0.50 0.51 0.64 0.08 0.806 0.839 0.070 Asp 0.61 0.53 0.45 0.59 0.08 0.639 0.615 0.063 Cys 0.15 0.13 0.10 0.15 0.02 0.673 0.460 0.081 Glu 0.78 0.68 0.56 0.71 0.09 0.421 0.737 0.068 Gly 1.81 1.30 1.80 1.98 0.34 0.487 0.340 0.056 Ser 0.44 0.37 0.34 0.43 0.06 0.840 0.741 0.084 Tyr 0.21 0.19 0.16 0.20 0.03 0.593 0.558 0.088 Total 11.18 8.71 13.41 14.33 1.99 0.191 0.491 0.149 Total amino acids 14.13 11.23 15.67 17.31 2.10 0.214 0.636 0.098 SEM, standard error of the means. www.animbiosci.org 1637 Lee et al (2021) Anim Biosci 34:1632-1642 Table 8. Standardized ileal digestibility (%) of amino acids in corn without or with 40 mL of 3 N HCl in the collection bag or the collection container1) HCl in bag − + p-value Item (%) SEM HCl in container − + − + Bag Container Bag×container Observations (n) 8 8 9 9 - - - - Indispensable amino acids Arg 89.0 85.2 92.5 95.5 6.0 0.410 0.909 0.303 His 86.1 81.6 87.4 87.9 2.5 0.251 0.311 0.210 Ile 83.0 81.5 82.2 86.0 2.2 0.521 0.447 0.099 Leu 88.9 87.8 88.9 90.6 1.4 0.447 0.781 0.214 Lys 80.3 81.5 76.4 81.7 3.3 0.676 0.163 0.353 Met 86.9 86.5 86.9 89.5 1.4 0.392 0.293 0.166 Phe 86.4 85.2 86.3 88.6 1.7 0.422 0.668 0.194 Thr 78.6 76.2 76.6 82.6 3.0 0.575 0.407 0.063 Trp 86.4 84.6 85.2 87.9 2.3 0.723 0.799 0.208 Val 80.1 78.8 80.3 83.8 2.5 0.443 0.498 0.155 Total 85.1 83.5 85.0 88.0 2.1 0.448 0.618 0.112 Dispensable amino acids Ala 86.0 85.2 85.8 89.8 2.0 0.408 0.281 0.111 Asp 81.3 79.6 79.8 84.0 2.4 0.654 0.458 0.097 Cys 79.6 78.6 79.5 83.4 2.3 0.439 0.415 0.161 Glu 87.4 86.8 87.5 89.4 1.5 0.509 0.544 0.235 Gly 82.6 71.4 82.0 88.3 8.8 0.515 0.567 0.055 Ser 85.4 83.4 84.5 87.7 2.0 0.512 0.674 0.094 Tyr 81.3 80.2 82.1 84.7 2.5 0.419 0.664 0.335 Total 83.2 81.5 89.0 90.9 4.9 0.315 0.976 0.472 Total amino acids 84.6 82.8 88.0 90.2 3.3 0.277 0.932 0.301 SEM, standard error of the means. 1) Within treatment values for standardized ileal digestibility were calculated by correcting apparent ileal digestibility values for basal ileal endogenous losses determined from ileal digesta that was treated or not treated with HCl in a similar way. middlings was in agreement with previous data [16], but the of AA, ileal digestibility was increased, decreased, or remained SID of Lys in wheat middlings was greater than reported the same if microbial fermentation affects the collected ileal [16]. Lysine is usually the AA that is most susceptible to heat digesta. damage that may take place during production because Lys Because microbial fermentation also occurs in the small has an amino group in the side chain [17]. The fact that the intestine of pigs [2,3,19], stopping microbial action after col- SID of Lys in wheat middlings used in this experiment was lection of ileal digesta is necessary. By storing ileal digesta in greater compared with previous values indicates that heat a –20°C freezer immediately after collection, microbial action damage during processing was minimal. Greater standard can be prevented [20,21]. In most AA digestibility experi- error of the means values for the AID and SID of AA were ments the collected ileal digesta are stored at –20°C to prevent observed in wheat middlings compared with corn and SBM. microbial degradation of AA. However, it has been suggested Wheat middlings contain more fiber than corn and SBM, that adding acid to collection bags [4,5] or collection con- and it is possible that greater concentration of fiber increases tainers [6,7] may reduce pH and, therefore, stop fermentation variations in digestibility values. Recovery of the indigestible in the digesta [22,23], which theoretically will result in changed marker can also be affected by fiber [18], which may result SID of AA. Both formic acid (HCOOH) and HCl have been in greater variations. used in previous experiments whereas only HCl was used in Dietary protein is digested and absorbed as free AA or this experiment. Formic acid is less acidic compared with small peptides in the small intestine of pigs, but undigested HCl because of the lower acid dissociation constant, which and unabsorbed N and AA are fermented in the hind-gut. may result in a reduced ability to reduce pH in the ileal di- Addition of antibiotics to diets fed to pigs reduces N fermen- gesta. It is, therefore, unlikely that formic acid would produce tation [19], which indicates that prevention of microbial results that are different from those observed in the present fermentation in ileal digesta also prevents the change in AA experiment with HCl. composition of the ileal digesta of pigs. Because AA are de- However, in some experiments, no attempt to reduce pH graded, synthesized, or both degraded and synthesized by is made because it is assumed that the rapid reduction in microbial fermentation [12], it was expected that depending temperature after collection that is a consequence of storing 1638 www.animbiosci.org Lee et al (2021) Anim Biosci 34:1632-1642 Table 9. Standardized ileal digestibility (%) of amino acids in soybean meal without or with 40 mL of 3 N HCl in the collection bag or the collection container1) HCl in bag − + p-value Item (%) SEM HCl in container − + − + Bag Container Bag×container Observations (n) 9 8 9 9 - - - - Indispensable amino acids Arg 97.0a 95.0b 96.1ab 97.3ab 1.1 0.637 0.520 0.010 His 88.5 89.0 87.8 88.4 1.5 0.756 0.465 0.932 Ile 85.2 85.5 85.1 87.2 1.8 0.762 0.172 0.329 Leu 85.4 85.4 85.0 86.9 1.8 0.819 0.295 0.293 Lys 86.2 86.8 86.4 88.5 1.6 0.671 0.122 0.353 Met 87.2 87.7 86.2 88.8 1.6 0.990 0.041 0.150 Phe 85.5 85.6 85.4 87.1 1.8 0.787 0.273 0.355 Thr 82.9 81.4 80.7 83.5 2.2 0.972 0.618 0.101 Trp 91.4 90.6 90.6 91.6 1.2 0.958 0.935 0.264 Val 82.2 81.7 81.2 84.0 2.5 0.851 0.354 0.193 Total 87.2 86.8 86.6 88.5 1.5 0.803 0.331 0.163 Dispensable amino acids Ala 80.6 80.5 80.9 85.5 3.0 0.527 0.180 0.161 Asp 81.6 82.6 82.9 84.3 1.9 0.561 0.292 0.863 Cys 75.8 75.6 75.1 78.0 3.2 0.841 0.491 0.433 Glu 82.1 84.0 83.9 85.0 2.3 0.653 0.286 0.761 Gly 81.3ab 75.9b 84.7ab 91.0a 4.3 0.116 0.850 0.043 Ser 88.6a 85.6b 85.6ab 87.7ab 1.1 0.739 0.630 0.008 Tyr 87.7 86.3 86.2 87.9 1.8 0.989 0.850 0.110 Total 84.6 83.7 88.3 91.4 2.4 0.084 0.469 0.185 Total amino acids 86.1 85.4 87.7 90.3 1.8 0.172 0.422 0.170 SEM, standard error of the means. 1) Within treatment values for standardized ileal digestibility were calculated by correcting apparent ileal digestibility values for basal ileal endogenous losses determined from ileal digesta that was treated or not treated with HCl in a similar way. a,b Within a row, means without a common superscript differ (p < 0.05). digesta at –20°C is sufficient to prevent microbial degrada- on the SID of any indispensable AA in wheat middlings, the tion of AA [24-27]. There are no data demonstrating the differences observed for AID of Met is likely not of any con- necessity of adding acids to digesta collection bags or collec- sequence in practical diet formulation. tion containers and it is unlikely that acids added to collection The tendency for an interaction between adding HCl to bags will impact fermentation because no mixing of digesta collection bags and to collection containers that was observed and acids takes place in the bags. However, if acids are added for the basal endogenous losses of Lys and all dispensable to collection containers, acids can be mixed with digesta, but AA may be a result of a larger proportion of the AA reach- the observation that adding HCl to collection containers did ing the hindgut of pigs fed the N-free diet being of microbial not impact digestibility indicates that reduction of pH is not origin compared with pigs fed the other diets. Although the necessary to prevent fermentation. This is in agreement with majority of microbial action takes place in the large intestine, data indicating that adjustment of pH by adding HCl to there is some microbial activity in the small intestine [3], thawed ileal digesta before drying did not affect AID of AA which may impact small intestinal digesta differently if an in diets [28]. The present results indicating no impact of add- N-free and low fiber diet is provided rather than a diet con- ing HCl to ileal digesta before freezing, therefore, complement taining AA and fiber at greater levels. However, research is the previous data. The observation that the AID of Met and needed to verify this hypothesis. total dispensable AA was increased by adding HCl to collec- It is possible that changing collection bags at least every tion bags or containers may be a consequence of the fact that 30 min or whenever they are full contributed to the lack of microbes in the intestinal tract prefer peptides or ammonia difference observed in this experiment because several hours as N rather than free AA [29]. It is, therefore, possible that are needed for degradation of AA by microbes [30-32]. The microbes produce more Met and dispensable AA, specifically observation that there were no effects of HCl in collection Gly, compared with other AA, which may result in an in- bags or containers on AID and SID of most AA in corn, SBM, crease in concentrations of Met and Gly in the ileal digesta and wheat middlings thus indicates that the rapid reduction with no HCl. However, because there was no impact of acids in temperature after collection is sufficient to prevent micro- www.animbiosci.org 1639 Lee et al (2021) Anim Biosci 34:1632-1642 Table 10. Standardized ileal digestibility (%) of amino acids in wheat middlings without or with 40 mL of 3 N HCl in the collection bag or the collection container1) HCl in bag − + p-value Item (%) SEM HCl in container − + − + Bag Container Bag×container Observations (n) 8 9 8 9 - - - - Indispensable amino acids Arg 84.0 82.0 84.3 94.7 6.1 0.412 0.330 0.161 His 77.6 78.6 81.8 83.5 3.2 0.307 0.497 0.861 Ile 73.5 72.9 74.1 75.9 2.7 0.530 0.814 0.649 Leu 76.5 75.9 78.5 79.9 2.2 0.195 0.875 0.649 Lys 78.1 77.2 75.8 78.5 3.0 0.909 0.627 0.335 Met 76.1 74.7 79.4 80.2 2.1 0.116 0.865 0.520 Phe 77.0 75.8 78.8 80.1 2.2 0.190 0.977 0.551 Thr 68.7 66.6 68.6 71.5 4.5 0.598 0.933 0.586 Trp 77.0 76.2 77.9 81.5 3.3 0.420 0.650 0.466 Val 71.2 71.2 73.1 74.9 2.6 0.303 0.730 0.730 Total 76.5 75.4 77.4 80.8 2.8 0.333 0.659 0.381 Dispensable amino acids Ala 61.4 64.7 62.3 73.3 5.0 0.387 0.128 0.401 Asp 72.5 71.0 70.6 75.3 3.3 0.727 0.616 0.338 Cys 75.0 74.0 74.1 79.0 2.9 0.515 0.502 0.316 Glu 85.5 85.2 84.7 87.3 1.5 0.703 0.413 0.308 Gly 50.8 70.7 49.7 77.0 16.1 0.875 0.160 0.821 Ser 76.3 74.0 75.3 79.3 3.7 0.601 0.805 0.384 Tyr 77.2 75.9 77.0 79.0 3.0 0.655 0.898 0.570 Total 58.7 66.6 55.4 81.8 12.8 0.717 0.070 0.313 Total amino acids 67.6 71.6 66.2 82.1 8.0 0.652 0.102 0.315 SEM, standard error of the means. 1) Within treatment values for standardized ileal digestibility were calculated by correcting apparent ileal digestibility values for basal ileal endogenous losses determined from ileal digesta that was treated or not treated with HCl in a similar way. bial degradation of AA. The observation that endogenous Results and their practical applications. A review. Livest Prod losses of AA were also not affected by acids in collection Sci 1986;15:367-88. https://doi.org/10.1016/0301-6226(86)90 bags or containers further support the hypothesis that it is 076-X not necessary to use HCl in AA digestibility experiments. 2. Jaworski NW, Stein HH. Disappearance of nutrients and energy in the stomach and small intestine, cecum, and colon CONCLUSION of pigs fed corn-soybean meal diets containing distillers dried grains with solubles, wheat middlings, or soybean If digesta are stored at –20°C immediately after collection, hulls. J Anim Sci 2017;95:727-39. https://doi.org/10.2527/ there are no clear advantages of adding HCl to digesta col- jas.2016.0752 lection bags or collection containers. Values for the basal 3. Miner-Williams W, Moughan PJ, Fuller MF. Endogenous endogenous losses of AA and the AID and SID of most AA components of digesta protein from the terminal ileum of are not changed by HCl and it is, therefore, most practical to pigs fed a casein-based diet. J Agric Food Chem 2009;57: avoid adding HCl to collection bags or collection containers. 2072-8. https://doi.org/10.1021/jf8023886 4. Mariscal-Landin G, Reis de Souza TC, Avalos MA. Ileal CONFLICT OF INTEREST amino acids digestibility of sorghum in weaned piglets and growing pigs. Animal 2010;4:1341-8. https://doi.org/10.1017/ We certify that there is no conflict of interest with any financial S175 1731110000273 organization regarding the material discussed in the manu- 5. Park CS, Helmbrecht A, Htoo JK, Adeola O. Comparison of script. Navarro DMDL is an employee of Hamlet Protein Inc. amino acid digestibility in full-fat soybean, two soybean meals, and peanut flour between broiler chickens and growing pigs. 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