M. Z. Fan*,2 and W. C. Sauer. Introduction

Transcription

1 Determination of true ileal amino acid digestibility and the endogenous amino acid outputs associated with barley samples for growing-finishing pigs by the regression analysis technique 1 M. Z. Fan*,2 and W. C. Sauer *Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1 and Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5 ABSTRACT: This study was conducted to determine true ileal AA digestibility coefficients and the endogenous AA outputs associated with barley samples for growing-finishing pigs using the regression analysis technique with dual digestibility markers. Six barrows, with 30.5 and 58.6 kg average initial and final BW, were fitted with a simple T-cannula at the distal ileum and fed six barley-based diets at close to ad libitum feed intake according to a 6 6 Latin square design. The six diets contained 97% of six barley samples varying from low to high in CP and AA contents (8.5, 9.2, 9.8, 11.5, 12.6, and 15.6% CP, respectively, on DM basis). The dietary NDF content ranged from 16.8 to 23.8% on DM basis. Chromic oxide (Cr 2 O 3 ) and acid-insoluble ash (AIA) were used as digestibility markers. Each experimental period lasted 7 d. Ileal digesta were collected, at 2-h intervals, for a total of 24 h duringd6and 7. There were linear relationships (P < 0.01) between dietary contents of apparent ileally digestible and total CP and AA as determined by using either Cr 2 O 3 or AIA as a digestibility marker. The use of Cr 2 O 3 vs AIA affected (P < 0.01) the determination of true ileal AA digestibility coefficients and the endogenous CP and AA outputs. However, there were no differences (P > 0.01) in the true ileal AA digestibility coefficients in barley samples between this study and the average values reported in the literature. The endogenous CP and AA outputs determined in this study were higher (P < 0.01) than reported values (35.1 ± 3.0 vs 14.7 ± 1.1 g CP/kg DMI). It is concluded that dual digestibility markers should be used to measure true ileal AA digestibility coefficients and endogenous AA outputs when dietary fiber content is high and the ileal digesta is collected through a simple T-cannula in the pig. True rather than apparent ileal AA digestibility coefficients determined in barley samples should be used in diet formulation for swine. The gastrointestinal endogenous AA secretion, recycling, and output losses are important in whole-body AA utilization and homeostasis, especially when fiber-enriched diets are fed to growing-finishing pigs. Key Words: Amino Acids, Barley, Digestibility, Pigs 2002 American Society of Animal Science. All rights reserved. J. Anim. Sci : Introduction There is considerable variation in the apparent ileal CP and AA digestibility coefficients among various barley samples (Buraczewska et al., 1987; Sauer and Ozimek, 1986). Because of differences in the endogenous 1 We are very grateful to B. Tchir and C. Gorsak for their assistance with animal surgery and to R. Van Weelden, R. McDaniel, M. Phelps, G. Francisco, and K. Schuchart (Heartland Lysine Inc., Chicago, IL) for the analyses of amino acids. Financial support provided by the Natural Sciences and Engineering Research Council of Canada; the Alberta Agricultural Research Institute, Halchemix Canada Inc., Toronto, ON; and Heartland Lysine Inc., Chicago, IL, is gratefully acknowledged. 2 Correspondence: Room 250, #70 Animal Science/Nutrition Building (phone: (519) , ext. 3656; fax: (519) ; mfan@uoguelph.ca). Received June 20, Accepted January 25, CP and AA contributions, methodological factors such as determination methods and differences in CP and AA contents between assay diets are primarily responsible for this variation (Fan et al., 1994; Fan and Sauer, 1995). True ileal CP and AA digestibility coefficients in barley samples have been previously reported (Sauer et al., 1974, 1981; de Lange et al., 1990). However, in the aforementioned studies, true AA digestibility coefficients were obtained by assuming constant AA profile of endogenous gut proteins determined from pigs fed protein-free diets. It is not clear how differences in the texture of test feed ingredients, such as different barley samples, affect the endogenous AA contributions and true AA digestibility coefficients. On the contrary, the regression analysis technique will allow the determination of true ileal AA digestibility and the endogenous 1593

2 1594 Fan and Sauer AA outputs in the presence of assay ingredients (Taverner et al., 1981a). An additional issue facing the determination of true ileal AA digestibility coefficients and the endogenous AA outputs in barley samples is the use of a single digestibility marker, e.g., chromic oxide (Cr 2 O 3 ), in combination with spot sampling of digesta from a simple T-cannula (Köhler et al., 1990). Previous studies have shown that the use of a single marker can result in misleading ileal nutrient digestibility coefficients in diets high in fiber content when ileal digesta samples are obtained through a T-cannula (Köhler et al., 1990). Under this context, this study was conducted to determine true ileal AA digestibility coefficients and the endogenous AA outputs associated with barley samples for growing-finishing pigs using the regression analysis technique. A related objective was to examine the effects of using dual digestibility markers, Cr 2 O 3 and acid-insoluble ash (AIA), on the determination of true ileal AA digestibility coefficients and the endogenous AA outputs in pigs fed barley-based diets. Principles of Extrapolation Materials and Methods True ileal CP and AA digestibility coefficients and endogenous CP and AA outputs can be determined by the regression analysis technique according to Furuya and Kaji (1986) and Fan et al. (1995) as described in Eq. [1]: A Ai = A E + D T A Di [1] where A Ai = apparent ileal digestible contents of CP and AA in the i th assay diet (g/kg DM); i = the number of assay diets; A E = levels of CP and AA of the endogenous origin in ileal digesta (g/kg DMI); D T = true ileal digestibility coefficients of CP and AA in test ingredients (decimal percentage); and A Di = total CP and AA contents in the i th assay diet (g/kg DM). Equation [1] represents a simple linear regression model, in which A Ai and A Di are dependent and independent variables, respectively (Fan et al., 1995). Animals, Diets, and Design Six barrows (Lacombe Yorkshire), with an average initial BW of 30.5 kg, were surgically fitted with a simple T-cannula at the distal ileum according to procedures adapted from Sauer et al. (1983). After surgery, the barrows were individually housed in stainless steel metabolic crates in a temperature-controlled barn (20 22 C). During a 14-d recovery period, the barrows were fed a 16% CP grower diet. A detailed description of preand postoperative care was previously presented by Sauer et al. (1983). Following recovery, the barrows were fed one of the six experimental diets according to 6 6 Latin square design. The barrows were fed twice daily, at 0800 and Feed intake was 1,600 g/d during Period 1 and was increased by 160 g/d consecutively for the other five periods. Water was freely available from a low-pressure drinking nipple. At the conclusion of the experiment, the barrows, with an average final BW 58.6 kg, were electrically stunned before killing, bled out, and dissected to determine whether cannulation had caused intestinal abnormalities. Six barley samples selected for these studies were obtained from three barley cultivars and from three locations, namely, cv. Hurrington from Saskatchewan Wheat Pool, cv. Argyle from Saskatchewan Wheat Pool, cv. Hurrington from Alberta United Grain Growers, cv. Bonanza from Saskatchewan Wheat Pool, cv. Hurrington Alberta Wheat Pool, and cv. Bonanza from Alberta United Grain Growers. Therefore, differences among the barley samples reflect the combined effects of cultivars, environmental factor, and growing conditions. Six barley-based diets contained 97% of the respective barley samples that provided the sole dietary AA. Vitamins and minerals were supplemented according to NRC (1988) standards because the animal trial was conducted before the publication of the NRC (1998). Chromic oxide (0.30%; Fisher Scientific, Fair Lawn, NJ) was included in the diets as a liquid-phase digestibility marker. The barley samples were ground through a 4.8-mm mesh screen prior to diet incorporation. Each experimental period lasted 7 d. Ileal digesta were collected for a total of 24 h: from 0800 to 1000 on d 6 and every other 2 h thereafter until 0800 on d 7 and from 1000 to 1200 on d 7 and every other 2 h thereafter until 0800 h on d 8. Ileal digesta were collected in soft plastic tubing (length, 15 cm; i.d., 2.5 cm), which was attached to the barrel of the cannula with a Velcro-elastic strap. The tubing contained about 10 ml of a solution of formic acid (10%, vol/vol) to minimize microbial activity. The tubing was removed and replaced as soon as it was partially filled with digesta. Digesta were immediately frozen at 20 C following collection. The experimental proposal, surgical procedures, and procedures for care and treatment of the barrows were reviewed and approved by the Faculty of Agriculture and Forestry Animal Care Committee of the University of Alberta. The barrows used in this experiment were cared for in accordance with the guidelines established by CCAC (1980). Chemical Analyses After the conclusion of the animal trial, digesta samples were freeze-dried, pooled within barrow and period for the same dietary treatment, ground through a 0.8- mm mesh screen in a Wiley mill (Arthur H. Thomas Co., Philadelphia, PA), and mixed before analyses. The barley samples and the diets were ground similarly. Analyses for DM and CP were carried out according to AOAC (1984) methods. Analysis for NDF was carried out according to principles outlined by Goering and Van

3 True ileal amino acid digestibility in barley 1595 Table 1. Dietary contents (g/kg DM) of total crude protein, neutral-detergent fiber, and amino acids in the barley-based diets for growing-finishing pigs Experimental diet a Item B1 B2 B3 B4 B5 B6 Dry matter Crude protein Neutral-detergent fiber Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Alanine Aspartic acid Cysteine Glutamic acid Glycine Proline Serine Tyrosine a The six barley-based diets (B1 to B6) contained the following barley samples. B1, cv. Hurrington from Saskatchewan Wheat Pool; B2, cv. Argyle Saskatchewan Wheat Pool; B3, cv. Hurrington from Alberta United Grain Growers; B4, cv. Bonanza from Saskatchewan Wheat Pool; B5, cv. Hurrington Alberta Wheat Pool; and B6, cv. Bonanza from Alberta United Grain Growers. Soest (1970). Chromic oxide content was determined according to the procedure of Fenton and Fenton (1979). Acid-insoluble ash was measured according to McCanthy et al. (1974) and Van Keulen and Young (1977). For AA analyses, with the exception of the sulfurcontaining AA and tryptophan, the samples were hydrolyzed with 6 M HCl at 110 C for 24 h, and AA were derivatized as ninhydrin-positive compounds and detected colorimetrically according to procedures adapted from Mason et al. (1980) using an automatic AA analyzer (Beckman model 6300, Beckman Inst., Inc., Palo Alto, CA.). Methionine and cysteine contents were determined as methionine sulfone and cysteic acid after oxidation with performic acid; the oxidation process was carried out according to AOAC (1984). The oxidized samples were then hydrolyzed and analyzed in the same manner as the samples that were not oxidized. Tryptophan analysis was carried out according to the procedure described by Jones et al. (1981). Calculations and Statistical Analyses The apparent ileal CP and AA digestibility coefficients in the diets (and also in the barley samples) were determined according to Eq. [2]: D Ai = 100 [(I Di A Ii )/(I Ii A Di )] 100 [2] where D Ai = apparent ileal CP and AA digestibility coefficients in the i th assay diet (%); I Di = contents of digestibility markers, Cr 2 O 3 and AIA, in the i th assay diet (%, on as-fed basis); A Ii = contents of CP and AA in the ileal digesta of the i th assay diet (%, on as-fed basis); I Ii = contents of digestibility markers, Cr 2 O 3 and AIA, in the ileal digesta of the i th assay diet (%, on as-fed basis); and A Di = contents of CP and AA in the ith assay diet (%, on as-fed basis). The apparent ileal digestible CP and AA contents in the barley-based diets were calculated according to Eq. [3]: ADi = D Ai AAi [3] where ADi = apparent ileal digestible contents of CP and AA in the i th assay diet (g/kg DM); i = the number of assay diets; D Ai = apparent ileal CP and AA digestibility coefficients in the i th assay diet (%); and AAi = total CP and AA contents in the i th assay diet (g/kg DM). If the true ileal digestibility coefficients of CP and AA in assay diets are obtained with the regression analysis, the levels of the endogenous CP and AA in the ileal digesta can also be determined according to Eq. [4]: A E = (AAi D T )/100 AD i [4]

4 1596 Fan and Sauer Table 2. Dietary contents (g/kg DM) of apparent ileal digestible crude protein and amino acids in the barley-based diets for growing-finishing pigs as determined by using chromic oxide as a digestibility marker Experimental diet a Item B1 B2 B3 B4 B5 B6 SEM b Crude protein c d e f f g 2.55 Arginine 0.48 c 0.77 c 2.83 d 5.06 e 6.88 f 8.31 f 0.50 Histidine 1.17 c 1.07 c 0.98 c 1.49 d 1.73 e 2.19 f 0.05 Isoleucine 0.97 c 1.21 cd 1.42 d 1.95 e 2.07 e 3.02 f 0.08 Leucine 2.31 c 2.07 c 3.29 d 4.17 e 4.82 f 6.43 g 0.15 Lysine 0.93 c 1.05 cd 1.10 cd 1.48 de 1.66 e 2.24 f 0.13 Methionine 0.75 c 0.96 d 0.96 d 1.06 d 1.30 e 1.71 f 0.05 Phenylalanine 1.08 c 1.72 d 2.09 e 3.04 f 3.49 g 4.76 h 0.11 Threonine 0.97 c 0.85 c 1.04 c 1.52 d 1.56 d 2.66 e 0.13 Tryptophan 0.19 c 0.33 d 0.45 e 0.68 f 0.83 g 1.00 h 0.03 Valine 2.07 c 2.41 d 2.54 d 3.34 e 3.70 f 5.14 g 0.11 Alanine 0.52 c 0.67 c 0.75 c 1.29 d 1.27 d 2.46 e 0.13 Aspartic acid 1.23 c 1.65 c 1.82 c 2.59 d 2.57 d 4.21 e 0.19 Cysteine 1.74 c 1.66 c 1.48 d 2.38 e 2.71 f 2.98 g 0.06 Glutamic acid 7.83 c d e f g h 0.68 Glycine 0.04 c 0.06 c 0.05 c 0.69 d 0.72 d 1.86 e 0.16 Proline 3.08 c 2.52 c 3.76 c 6.70 d 3.76 c 6.42 d 0.42 Serine 1.08 c 1.26 c 1.40 c 2.12 d 2.28 d 3.44 e 0.11 Tyrosine 0.01 c 0.52 d 0.45 d 0.38 d 1.03 e 1.67 f 0.08 a The six barley-based diets (B1 to B6) contained the following barley samples. B1, cv. Hurrington from Saskatchewan Wheat Pool; B2, cv. Argyle Saskatchewan Wheat Pool; B3, cv. Hurrington from Alberta United Grain Growers; B4, cv. Bonanza from Saskatchewan Wheat Pool; B5, cv. Hurrington Alberta Wheat Pool; and B6, cv. Bonanza from Alberta United Grain Growers. b Pooled standard error of the means (n = 6). c,d,e,f,g,h Means in the same row followed by different superscript letters differ (P < 0.01). where A E = the level of CP and AA of the endogenous origin in ileal digesta (g/kg DMI); AAi = total CP and AA contents in the i th assay diet (g/kg DM); i = the number of assay diets; D T = true ileal digestibility coefficients of CP and AA in the test ingredients (decimal percentage); and AD i = apparent ileal digestible CP and AA contents in the i th assay diet (g/kg DM). Periods and animals were the controlled factors in the 6 6 Latin square design. The contents of apparent ileal digestible CP and AA and true ileal digestibility coefficients of CP and AA were first subjected to ANOVA for a 6 6 Latin square design. The ANOVA was carried out using the GLM procedures of SAS (SAS Inst. Inc., Cary, NC). To determine the levels of endogenous CP and AA in ileal digesta by the regression analysis, simple linear relationships between dietary contents of apparent ileal digestible and dietary contents of total CP and AA were established. The comparisons between the two digestibility markers in determining true ileal CP and AA digestibility coefficients and the levels of endogenous CP and AA in ileal digesta were conducted according to the pooled t-test (Byrkit, 1987). Analyses of simple linear regression were carried out using the GLM procedures of SAS. Results Dietary NDF content of the six diets ranged from 16.8 to 23.8% on DM basis (Table 1). Because the same dietary inclusion level of barley (97%) was used for all the diets, and the barley samples were different in CP and AA contents, CP and AA contents increased from low to high between diets B1 and B6 (Table 1). The contents of apparent ileal digestible CP and AA in the diets were determined according to Eq. [2] and [3] by using Cr 2 O 3 and AIA as digestibility markers, respectively. Because the total and the apparent digestible contents of CP and AA were not equally spaced between diets, differences in the apparent ileal digestible contents of CP and AA among the six diets were not statistically analyzed by the orthogonal polynomial procedure, but by multiple comparison. As determined by using Cr 2 O 3 and AIA as digestibility markers, there were differences (P < 0.01) in the contents of apparent ileal digestible CP and AA among the six diets (Tables 2 and 3). There were linear relationships (P < 0.01) between the dietary contents of the apparent ileal digestible and the total CP and AA contents as determined by using Cr 2 O 3 and AIA as digestibility markers (Tables 4 and

5 True ileal amino acid digestibility in barley 1597 Table 3. Dietary contents (g/kg DM) of apparent ileal digestible crude protein and amino acids in the barley-based diets for growing-finishing pigs as determined by using the acid-insoluble ash as a digestibility marker Experimental diet a Item B1 B2 B3 B4 B5 B6 SEM b Crude protein c d de de e f 4.65 Arginine 0.68 c 1.46 cd 2.92 d 5.22 e 7.49 f 7.96 f 0.59 Histidine 1.22 cd 1.31 cd 1.14 c 1.53 d 1.86 e 2.11 e 0.09 Isoleucine 1.07 c 1.62 d 1.70 d 2.02 de 2.32 e 2.90 f 0.13 Leucine 2.52 c 3.49 d 3.83 d 4.32 d 5.29 e 6.19 f 0.25 Lysine 1.05 c 1.63 cd 1.46 cd 1.58 cd 1.93 d 2.08 d 0.19 Methionine 0.80 c 1.17 d 1.10 d 1.10 d 1.42 e 1.65 f 0.07 Phenylalanine 1.21 c 2.15 d 2.41 d 3.12 e 3.79 f 4.60 g 0.17 Threonine 1.11 c 1.46 c 1.45 c 1.63 c 1.87 c 2.47 d 0.20 Tryptophan 0.23 c 0.50 d 0.57 d 0.71 de 0.93 e 0.94 e 0.08 Valine 2.22 c 3.06 d 2.97 d 3.56 de 4.06 e 4.95 f 0.20 Alanine 0.66 c 1.32 cd 1.17 cd 1.40 cd 1.57 d 2.29 e 0.20 Aspartic acid 1.43 c 2.62 d 2.44 d 2.76 d 3.03 d 3.94 e 0.29 Cysteine 1.79 c 1.66 c 1.48 d 2.38 e 2.71 f 2.98 g 0.05 Glutamic acid 8.40 c d d e f g 0.89 Glycine 0.13 c 0.80 cd 0.46 cd 0.81 cd 1.14 cd 1.64 d 0.32 Proline 0.18 c 3.36 cd 3.09 cd 5.60 d 7.17 de e 1.15 Serine 1.22 c 1.82 cd 1.79 cd 2.22 cd 2.58 e 3.26 f 0.19 Tyrosine 0.07 c 0.79 d 0.66 d 0.44 d 1.20 e 1.58 f 0.12 a The six barley-based diets (B1 to B6) contained the following barley samples. B1, cv. Hurrington from Saskatchewan Wheat Pool; B2, cv. Argyle Saskatchewan Wheat Pool; B3, cv. Hurrington from Alberta United Grain Growers; B4, cv. Bonanza from Saskatchewan Wheat Pool; B5, cv. Hurrington Alberta Wheat Pool; and B6, cv. Bonanza from Alberta United Grain Growers. b Pooled standard error of the means (n = 6). c,d,e,f,g Means in the same row followed by different superscript letters differ (P < 0.01). 5). It is obvious that differences in NDF contents between diets (16.8 to 23.8%) did not affect these linear relationships, as presented in Tables 4 and 5; otherwise, these linear relationships would be insignificant. According to Eq. [1], the existence of linear relationships between the contents of total and apparent ileal digestible CP and AA in the barley-based diets suggests that true ileal CP and AA digestibility coefficients in the barley samples can be directly determined from the regression analysis. Slopes of the linear regression equations, as presented in Tables 4 and 5, provide the directly determined true CP and AA digestibility coefficients. There were differences (P < 0.01) in the true ileal CP and AA digestibility coefficients between the digestibility markers, Cr 2 O 3 and AIA. The true ileal CP and AA digestibility coefficients as determined with Cr 2 O 3 were considerably higher (P < 0.01) than the values measured by using AIA. Assuming equal mass distribution of both liquid and solid phases of ileal digesta, the true ileal CP and AA digestibility coefficients in the barley samples were represented by averaging the values determined with Cr 2 O 3 and AIA markers (Table 6). According to Eq. [1], intercepts of the linear equations, as presented in Table 7, provide the direct estimates of the corresponding endogenous CP and AA outputs in the ileal digesta. The intercepts of the linear regression equations, as determined by using Cr 2 O 3, were all different (P < 0.05) from zero; thus, these values were the estimated endogenous output values. In the situation of using AIA as a digestibility marker, the intercepts of linear regression equations were not different (P < 0.05) from zero for His, Lys, Thr, and Asp; thus, the endogenous outputs in the ileal digesta for these AA were alternatively determined according to Eq. [4] by using the corresponding true AA digestibility coefficients obtained from the regression equations presented in the Table 5. As shown in Table 7, there were differences (P < 0.01) in the outputs (g/kg DMI) of the endogenous CP and AA between the two digestibility marker methods. The outputs of the endogenous CP and AA, as determined by using Cr 2 O 3, were considerably higher than the values obtained by using the AIA marker. Again, assuming equal mass distribution of both liquid and solid phases of ileal digesta, the values of endogenous CP and AA, pooled between the Cr 2 O 3 and the AIA methods, were considered to be the outputs of CP and AA associated with barley samples fed to growing-finishing pigs (Table 7). The true ileal CP and AA digestibility coefficients in the barley samples determined from this study were generally not different (P > 0.05) from the average true digestibility coefficients reported in the literature (Table 8). On the other hand, the endogenous CP and AA

6 1598 Fan and Sauer Table 4. Linear relationships between dietary contents of the apparent ileal digestible and total crude protein and amino acids in barley samples for growing-finishing pigs as determined by using chromic oxide as a digestibility marker Item Regression equation ab r 2 S yx c P-value d P-value e Crude protein y = x Arginine y = x Histidine y = x Isoleucine y = x Leucine y = x Lysine y = x Methionine y = x Phenylalanine y = x Threonine y = x Tryptophan y = x Valine y = x Alanine y = x Aspartic acid y = x Cysteine y = x Glutamic acid y = x Glycine y = x Proline y = x Serine y = x Tyrosine y = x a y = dietary contents of apparent ileal digestible amino acids in the barley-based diets (g/kg DM). b x = dietary contents of crude protein and amino acids in the barley-based diets (g/kg DM). c Standard error of estimate of the regression equations (n = 36). d The probabilities of significance for the intercepts of the regression equations. e The probabilities of significance for the slopes of the regression equations. Table 5. Linear relationships between dietary contents of the apparent ileal digestible and the total crude protein and amino acids in barley samples for growing-finishing pigs as determined by using the acid-insoluble ash as a digestibility marker Item Regression equation ab r 2 S yx c P-value d P-value e Crude protein y = x Arginine y = x Histidine f y = x Isoleucine y = x Leucine y = x Lysine f y = x Methionine y = x Phenylalanine y = x Threonine f y = x Tryptophan y = x Valine y = x Alanine y = x Aspartic acid f y = x Cysteine y = x Glutamic acid y = x Glycine y = x Proline y = x Serine y = x Tyrosine y = x a y = dietary contents of apparent ileal digestible amino acids in the barley-based diets (g/kg DM). b x = dietary contents of crude protein and amino acids in the barley-based diets (g/kg DM). c Standard error of estimate of the regression equations (n = 36). d The probabilities of significance for the intercepts of the regression equations. e The probabilities of significance for the slopes of the regression equations. f The intercepts of the regression equations were not significant (P > 0.05, n = 36).

7 True ileal amino acid digestibility in barley 1599 Table 6. Comparison of true ileal crude protein and amino acid digestibility coefficients a (%) in barley samples for growing-finishing pigs as determined by using chromic oxide (Cr 2 O 3 ) and the acid-insoluble ash (AIA) as digestibility markers Item Cr 2 O 3 method b AIA method c The pooled d No. of observations Crude protein 86.1 ± 8.0 e 70.4 ± 8.1 f 78.3 ± 2.7 Arginine 93.2 ± 6.4 e 87.7 ± 6.3 f 90.5 ± 2.1 Histidine 92.5 ± 7.7 e 75.1 ± 8.3 f 83.8 ± 2.7 Isoleucine 95.0 ± 7.5 e 77.7 ± 8.2 f 86.4 ± 2.6 Leucine 96.6 ± 7.8 e 80.8 ± 8.2 f 88.7 ± 2.7 Lysine 95.0 ± 14.1 e 59.1 ± 17.3 f 77.1 ± 5.3 Methionine ± 9.3 e 83.5 ± 10.5 f 92.3 ± 3.3 Phenylalanine 92.8 ± 6.1 e 82.8 ± 6.4 f 87.8 ± 2.1 Threonine 97.4 ± 11.4 e 63.8 ± 13.2 f 80.6 ± 4.1 Tryptophan 90.4 ± 6.0 e 76.5 ± 9.1 f 83.5 ± 2.6 Valine 97.3 ± 7.9 e 79.3 ± 8.6 f 88.3 ± 2.8 Alanine ± 13.2 e 71.0 ± 15.0 f 86.9 ± 4.7 Aspartic acid 94.7 ± 9.7 e 66.2 ± 12.2 f 80.5 ± 3.7 Cysteine 95.3 ± ± ± 3.2 Glutamic acid 96.5 ± 6.1 e 88.5 ± 6.0 f 92.5 ± 2.0 Glycine ± 18.1 e 75.3 ± 22.3 f 89.2 ± 6.8 Proline 84.4 ± 10.4 e 96.8 ± 17.5 f 90.6 ± 4.8 Serine 94.1 ± 8.6 e 72.7 ± 10.0 f 83.4 ± 3.1 Tyrosine 92.0 ± 7.7 e 82.5 ± 8.3 f 87.3 ± 2.7 a Means ± SEM. b Values determined by using chromic oxide as a digestibility marker. c Values determined by using acid-insoluble ash as a digestibility marker. d Obtained by averaging the values determined by the chromic oxide and the acid-insoluble ash methods. The pooled standard errors were calculated according to Byrkit (1987). e,f Means in the same row having different superscript letters differ (P < 0.01). outputs determined from this study were generally several-fold higher than (P < 0.01) the average values reported in the literature for growing-finishing pigs (Table 9). Based on the estimated efficiency (75%) of reabsorption of the endogenous CP and AA secretions in the pig reported in the literature (Souffrant, 1991), levels of total endogenous CP and AA secretions, recycling, and the distal ileal losses, as a percentage of the diets on as-fed basis, were calculated for the growing-finishing pigs fed the barley-based diets in this study (Table 10). Total secretion of the endogenous CP was estimated to be about 70 to 79% of the requirements in the growingfinishing pig recommended by NRC (1998). Except Lys and His, total endogenous secretions of the other essential AA were considerably higher than the requirement levels recommended by NRC (1998) (Table 10). The estimated amounts of the endogenous CP and AA recycled within the gastrointestinal tract were, for most AA, close to the AA requirement levels recommended by NRC (1998) for growing pigs and higher than the requirement levels recommended by NRC (1998) in finishing pigs (Table 10). The losses of the endogenous dispensable AA, as a percentage of the diets, on asfed basis, were relatively low for Cys and Tyr (0.09); intermediate for Ala, Asp, Glc, and Ser (0.15 to 0.27); and high ( 0.41) for Glu, including Gln and Pro (Table 10). Discussion One of the major objectives of this study was to determine true ileal CP and AA digestibility coefficients in the barley samples varying in NDF (20.6 to 25.4%, on DM basis) and CP (8.7 to 16.3% CP, on DM basis) contents. Contrary to the large variability in the apparent ileal CP and AA digestibility coefficients reported in the literature (Buraczewska et al., 1987; Sauer and Ozimek, 1986), there is relatively small variation in the true ileal CP and AA digestibility coefficients among the barley samples. This is largely due to the fact that relative endogenous CP and AA contributions, as a percentage of dietary CP and AA contents, are curvilinearly dependent on the dietary contents of CP and AA, as was previously reported (Furuya and Kaji, 1989; Fan et al., 1994). Furthermore, true ileal CP and AA digestibility coefficients are considerably higher than reported apparent ileal CP and AA digestibility coefficients in barley samples (NRC, 1998). Apparent ileal CP and AA digestibility coefficients are usually much lower in barley samples than the values reported in other cereal grains, such as corn and wheat (Sauer and Ozimek, 1986). However, there are relatively small differences in true ileal CP and AA digestibility coefficients between barley samples and other cereal grains (NRC, 1998). These are all likely due to the fact that the endogenous CP and AA losses associated with barley

8 1600 Fan and Sauer Table 7. Comparison of the endogenous outputs a (g/kg DMI) of crude protein and amino acids from the ileal digesta of growing-finishing pigs fed with barley samples determined by using chromic oxide (Cr 2 O 3 ) and the acid-insoluble ash (AIA) as digestibility markers Item Cr 2 O 3 method b AIA method c The pooled d No. of observations Crude protein 46.6 ± 9.0 f 23.6 ± 9.2 g 35.1 ± 3.0 Arginine 3.30 ± 0.54 f 2.54 ± 0.53 g 2.92 ± 0.18 Histidine e 0.88 ± 0.20 f 0.44 ± 0.04 g 0.66 ± 0.05 Isoleucine 1.68 ± 0.28 f 0.89 ± 0.30 g 1.29 ± 0.10 Leucine 3.31 ± 0.59 f 1.80 ± 0.63 g 2.56 ± 0.20 Lysine e 2.26 ± 0.55 f 0.87 ± 0.07 g 1.57 ± 0.13 Methionine 0.97 ± 0.19 f 0.52 ± 0.22 g 0.75 ± 0.07 Phenylalanine 1.71 ± 0.30 f 1.06 ± 0.32 g 1.39 ± 0.10 Threonine 2.50 ± 0.47 f 1.14 ± 0.08 g 1.82 ± 0.11 Tryptophan 0.66 ± 0.08 f 0.41 ± 0.13 g 0.54 ± 0.04 Valine 2.69 ± 0.48 f 1.33 ± 0.53 g 2.01 ± 0.17 Alanine 2.85 ± 0.52 f 1.36 ± 0.59 g 2.11 ± 0.19 Aspartic acid e 3.75 ± 0.63 f 1.92 ± 0.11 g 2.84 ± 0.15 Cysteine 1.01 ± ± ± 0.11 Glutamic acid 7.82 ± 1.56 f 5.07 ± 1.54 g 6.45 ± 0.52 Glycine 3.63 ± 0.74 f 2.27 ± 0.92 g 2.95 ± 0.28 Proline 3.56 ± 1.15 f 5.44 ± 1.95 g 4.50 ± 0.53 Serine 2.25 ± 0.39 f 1.08 ± 0.45 g 1.67 ± 0.14 Tyrosine 1.11 ± 0.16 f 0.81 ± 0.17 g 0.96 ± 0.06 a Means ± SEM. b Values determined by using chromic oxide as a digestibility marker. c Values determined by using acid-insoluble ash as a digestibility marker. d Obtained by averaging the values determined by the chromic oxide and the acid-insoluble ash methods. The pooled standard errors were calculated according to Byrkit (1987). e Values for acid-insoluble ash method of these amino acids were not derived directly from the equations presented in Table 5, but were calculated according to Eq. [4]. f,g Means in the same row followed by different superscript letters differ (P < 0.01). samples are considerably high, as demonstrated in this study (Table 9). The linear relationships between the contents of total and apparent ileal digestible CP and AA in the barley-based diets suggest that the barley samples, although different in CP, AA, and NDF contents, are not different in true ileal CP and AA digestibility coefficients. Therefore, true rather than apparent ileal AA digestibility coefficients in barley samples should be used in the diet formulation for swine. An important observation of this study was that the use of digestibility markers, Cr 2 O 3 and AIA, had considerable effects on the determination of true CP and AA digestibility coefficients and the endogenous CP and AA outputs. These results suggest that there was separation of the ileal digesta into liquid and particulate phases in pigs fed the barley-based diets high in fiber content. This was also suggested by Köhler et al. (1990) in studies with pigs fed fiber-enriched diets. It was also interesting to note that the true ileal CP and AA digestibility coefficients determined with Cr 2 O 3 as a digestibility marker were considerably higher than the values measured with the AIA marker (Table 6). This was likely due to the fact that Cr 2 O 3 represented the liquid phase of the digesta and contained soluble fractions of the barley samples presumably higher in CP and AA digestibility, whereas AIA labeled the cell wall components of the barley samples in the digesta that contained aleurone layers low in CP and AA digestibility values. Alternatively, the relatively high levels of the endogenous CP and AA as determined by Cr 2 O 3 in comparison with the AIA marker was most likely due to the fact that most of the endogenous materials were likely associated with the liquid phase of the ileal digesta. In contrast to our results, studies by Van Leeuwen et al. (1996) found no differences in the ileal nutrient digestibility coefficients between the Cr 2 O 3 and the AIA methods. This was likely due to the fact that fiber content in the diets of their studies were not very high (Van Leeuwen, 1996). Thus, it is concluded that dual digestibility markers such as Cr 2 O 3 and AIA should be used to measure true ileal CP and AA digestibility coefficients and the endogenous CP and AA outputs in pigs when fiber-enriched diets are fed and the ileal digesta is collected through a simple T-cannula. On the other hand, it is technically difficult to differentiate the amount of liquid and solid phases of materials associated with ileal digesta. Thus, the use of dual digestibility markers introduces the question of how to obtain representative nutrient digestibility and endogenous output values from the results measured by using two

9 True ileal amino acid digestibility in barley 1601 Table 8. Comparison of true ileal crude protein and amino acid digestibility coefficients a (%) in barley samples determined from this study with values reported in the literature for growing-finishing pigs Item Present study b Summary of literature values c No. of observations or studies 72 2 to 9 Crude protein 78.3 ± ± 1.3 Arginine 90.5 ± ± 0.9 Histidine 83.8 ± ± 1.8 Isoleucine 86.4 ± ± 1.9 Leucine 88.7 ± ± 1.1 Lysine 77.1 ± ± 3.2 Methionine 92.3 ± ± 2.6 Phenylalanine 87.8 ± ± 2.0 Threonine 80.6 ± ± 4.2 Tryptophan 83.5 ± ± 6.1 Valine 88.3 ± ± 3.0 Alanine 86.9 ± ± 2.2 Aspartic acid 80.5 ± ± 3.2 Cysteine 94.8 ± ± 7.5 Glutamic acid 92.5 ± ± 1.1 Glycine 89.2 ± ± 5.8 Proline 90.6 ± ± 8.9 Serine 83.4 ± ± 2.5 Tyrosine 87.3 ± ± 4.2 a Means ± SEM. b The pooled values between the chromic oxide and the acid-insoluble ash methods from Table 6. c Based on literature references on de Lange et al. (1990), Nyachoti et al. (1997b), Sauer et al. (1974), Sauer et al. (1981), and Taverner et al. (1981b). different digestibility markers. The calculations in our studies are based on the assumption that the liquid and solid phases of materials, especially CP and AA, are similarly distributed in ileal digesta. Theoretically speaking, it will be ideal to use dual digestibility markers, in conjunction with quantitative information on the distribution of the liquid and solid phases of materials in ileal digesta, to measure true ileal CP and AA digestibility coefficients and the endogenous CP and AA outputs in pigs, when fiber-enriched diets are fed and the ileal digesta is collected through a simple T-cannula. The determination of factors influencing the total endogenous CP and AA secretion, recycling, and output losses at the end of the ileum has been a hot topic in AA nutrition research for many years (Nyachoti et al., 1997a; Fuller and Reeds, 1998). The endogenous CP and AA output losses in the ileal digesta determined in this study were dramatically higher than the average values reported in the literature for growing-finishing pigs (Table 9). Several factors are known to be partially responsible for the large differences in the endogenous CP and AA outputs. These factors include determination methods and(or) techniques used (de Lange et al., 1990; Mariscal-Landi et al., 1995), body weight and physiological status (de Lange et al., 1989; Stein et al., 1999; Hess and Sève, 1999), DM intake levels (Furuya and Kaji, 1991; Stein et al., 1999; Hess and Sève, 1999), and dietary fiber levels and types (Sauer et al., 1977; Leterme et al., 1992; Mariscal-Landi et al., 1995). Major methods and(or) techniques for the determination of the endogenous CP and AA outputs include 15 N- and 15 N-amino acid dilution techniques, homoarginine technique, protein-free feeding, and the regression analysis method. A close examination of the literature results revealed that, although there were differences between methods and techniques, the differences were not very large, as summarized and reflected by standard error values associated with these values (Table 9). Thus, differences among various determination methods were not likely responsible for the large differences in the levels of endogenous CP and AA between this study and the average values previously reported in the literature (Table 9). Differences in BW were shown to have no effects on the levels and composition of the endogenous CP (Stein et al., 1999). Dramatic differences in feed intake below the ad libitum level affected the endogenous CP level and amino acid composition (Furuya and Kaji, 1991; Stein et al., 1999). However, when feed intake level was close the level of ad libitum, it had no effects on the endogenous CP and AA composition (Hess and Sève, 1999). Differences in dietary fiber content were shown to affect the levels of endogenous CP and AA in the ileal digesta; however, the magnitude of the effects was dependent on the level of fiber in the diets (Sauer et al., 1974; Furuya and Kaji, 1991; Taverner et al., 1981a). The dietary fiber contents of the most reported studies were between 5 and 15% in NDF (Table 9). In light of the considerable differences in NDF con-

10 1602 Fan and Sauer Table 9. Comparison of the endogenous outputs a (g/kg DMI) of crude protein and amino acids from the ileal digesta associated with barley samples from this study with values reported in the literature for growing-finishing pigs Item Present study b Summary of literature values c No. of observations or studies to 41 NDF contents in diets 16.8 to to 15% Crude protein 35.1 ± 3.0 d ± 1.11 e Arginine 2.92 ± 0.18 d 0.53 ± 0.04 e Histidine 0.62 ± 0.07 d 0.21 ± 0.01 e Isoleucine 1.29 ± 0.10 d 0.36 ± 0.03 e Leucine 2.56 ± 0.20 d 0.58 ± 0.04 e Lysine 1.46 ± 0.20 d 0.45 ± 0.04 e Methionine 0.75 ± 0.07 d 0.15 ± 0.02 e Phenylalanine 1.39 ± 0.10 d 0.41 ± 0.03 e Threonine 1.71 ± 0.17 d 0.64 ± 0.05 e Tryptophan 0.54 ± 0.04 d 0.19 ± 0.05 e Valine 2.01 ± 0.17 d 0.51 ± 0.04 e Alanine 2.11 ± 0.19 d 0.63 ± 0.05 e Aspartic acid 2.66 ± 0.24 d 0.90 ± 0.06 e Cysteine 0.99 ± 0.11 d 0.20 ± 0.02 e Glutamic acid 6.45 ± 0.52 d 1.12 ± 0.10 e Glycine 2.95 ± 0.28 d 1.32 ± 0.12 e Proline 4.50 ± 0.53 d 3.02 ± 0.40 e Serine 1.67 ± 0.14 d 0.63 ± 0.05 e Tyrosine 0.96 ± 0.06 d 0.52 ± 0.08 e a Means ± SEM. b The pooled values between the chromic oxide and the acid-insoluble ash methods from Table 6. c Based on literature references on Chung and Baker (1992), de Lange et al. (1990), Fan and Sauer (1995), Fan and Sauer (1997), Furuya and Kaji (1989), Furuya and Kaji (1991), Hess and Sève (1999), Leterme et al. (1990), Leterme et al. (1996), Sauer et al. (1977), Stein et al. (1999), Nyachoti et al. (1997b), Sauer et al. (1974), Souffrant (1991), and Taverner et al. (1981a). d,e Means in the same row followed by different superscript letters differ (P < 0.01). tent between this study (an average of 21.6% NDF) and previously reported studies (an average of 10% NDF), we speculate that high NDF content was primarily responsible for the considerably high levels of the endogenous CP and AA observed in this study. Potential mechanisms on how dietary fiber levels and types affect the endogenous CP and AA levels in the ileal digesta are not clear. Dietary fiber can induce changes in plasma levels of glucagon-like peptide and insulin through short-chain fatty acids by intestinal fermentation (Reimer and McBurney, 1996). Furthermore, glucagon-like peptide-2 and insulin are known to be involved in regulating protein metabolism (Burrin et al., 2000; Davis et al., 2001). Therefore, one of the possible explanations is that differences in fiber content may affect protein synthesis and degradation in the gastrointestinal tract. This, in turn, may lead to changes in the total sloughing of mucosal proteins into the intestinal lumen, resulting in differences in the levels of the endogenous CP and AA in the ileal digesta. However, a recent study by Nyachoti et al. (2000) did not support this hypothesis. It should be pointed out that two dietary factors were confounded in the design of their study, including the use of different protein sources as well as different levels of protein and AA between the two test diets (Nyachoti et al., 2000). Thus further studies should be conducted on this topic. In addition, high dietary fiber content may enhance the total level of various endogenous secretions, such as biliary, exocrine pancreatic, and intestinal secretions (Low, 1989) as well as mucosal cell migration kinetics (Jin et al., 1994). All these can contribute to the increased levels of the endogenous CP and AA in the ileal digesta associated with high dietary fiber content in the barley-fed pigs. Additionally, barley is high in its content of other non-starch polysaccharides, such as β-glucans (Graham et al., 1989; Li et al., 1996). It is conceivable that the barley-based diets in our studies were high in β-glucan content. It was reported that a high viscosity of digesta flow was associated with pigs fed barley-based diets (Li et al., 1996). High digesta viscosity was associated with increased intestinal mucosal fractional protein synthesis (Dänicke et al., 2000), which, in turn, may result in increased levels of the endogenous CP and AA in the ileal digesta. It can be concluded that high NDF and β-glucan contents in this study, as a result of high dietary inclusion level of barley, might have been responsible for the considerably high levels of the endogenous CP and AA in the ileal digesta. However, the increased levels of CP and AA outputs in the ileal digesta did not affect the true ileal CP and AA digestibility coefficients in the barley samples.

11 True ileal amino acid digestibility in barley 1603 Table 10. Estimated total endogenous crude protein and amino acid secretion into the digestive tract, recycling and re-absorption and outputs (% of diets, on as-fed basis) from the ileal digesta associated with barley samples for growing-finishing pigs Total endogenous Recycling and Items secretion a reabsorption b Distal ileal losses c No. of observations Crude protein ± ± ± 0.27 Arginine 1.05 ± ± ± 0.02 Histidine 0.22 ± ± ± 0.01 Isoleucine 0.46 ± ± ± 0.01 Leucine 0.92 ± ± ± 0.02 Lysine 0.53 ± ± ± 0.02 Methionine 0.27 ± ± ± 0.02 Phenylalanine 0.50 ± ± ± 0.01 Threonine 0.62 ± ± ± 0.02 Tryptophan 0.19 ± ± ± 0.01 Valine 0.72 ± ± ± 0.02 Alanine 0.76 ± ± ± 0.02 Aspartic acid 0.96 ± ± ± 0.06 Cysteine 0.36 ± ± ± 0.02 Glutamic acid 2.32 ± ± ± 0.05 Glycine 1.06 ± ± ± 0.03 Proline 1.62 ± ± ± 0.05 Serine 0.60 ± ± ± 0.01 Tyrosine 0.35 ± ± ± 0.01 a Total endogenous crude protein and amino acid secretion (% of diets) = [distal ileal output (g/kg of dry matter intake)/0.25] /1000, based on the estimated reabsorption rate of 75% at the terminal ileum of the total endogenous nitrogen secretion by Souffrant et al. (1986) and Souffrant (1991) and the average dietary dry matter content of 90% (NRC, 1998). b Recycling and reabsorption of the total endogenous crude protein and amino acid secretion (% of diets) = [distal ileal outputs (g/kg of dry matter intake)/0.25] /1000, based on the estimated reabsorption rate of 75% at the terminal ielum of the total endogenous nitrogen secretion by Souffrant (1991) and the average dietary dry matter content of 90% (NRC, 1998). c Distal ileal crude protein and amino acid losses (% of diet) = [distal ileal outputs (g/kg of dry matter intake) 100/1000] 0.90, based on the average dietary dry matter content of 90% (NRC, 1998). A piece of striking evidence from this study was that the calculated total endogenous CP and AA secretions in growing-finishing pigs fed the barley-based diets in most cases exceeded the requirements of CP and AA recommended by NRC (1998). The amounts of calculated endogenous CP and AA recycled within the gastrointestinal tract were considerably large (Table 10). Taken together, the total endogenous CP and AA secretions play a pivotal role in maintaining whole-body AA nutrition and homeostasis. Furthermore, the distal ileal output of the endogenous indispensable AA, as a percentage of the requirements recommended by NRC (1998), ranged from 15% for Lys to 80% for Arg in the 20- to 50-kg growing pig and from 20% for Lys to 110% for Arg in the 50- to 80-kg finishing pig. The traditional metabolic fates of indispensable AA utilization in the animal were for oxidative catabolism (Burrin and Reeds, 1997; Wu, 1998; Reeds and Burrin, 2000) and various biosyntheses (Burrin and Reeds, 1997). As demonstrated from this study and many other studies (Souffrant et al., 1996; Souffrant, 1991), the gastrointestinal endogenous loss is also one of the major routes of metabolic inefficiency. As can be expected, the role of the endogenous AA secretion, recycling, and loss can become quantitatively very important in whole-body AA utilization and homeostasis, especially when animals are under abnormal nutrition and physiological conditions, such as starving, suffering from enteric diseases, challenging by toxic compounds and antinutritive factors, such as tannins and legume lectins. Furthermore, the considerable endogenous losses of dispensable AA especially Asp including Asn, Glu including Gln, Gly and Pro suggest that a minimal level of dietary supply of dispensable AA is essential to optimize whole-body growth and protein deposition (Lenis et al., 1999). Oxidative catabolism (Windmueller and Spaeth, 1980; Wu, 1998; Burrin and Reeds, 1997), biosynthesis (Burrin and Reeds, 1997; Wu, 1998), and the gastrointestinal endogenous losses, as demonstrated from this study, are also the major routes of dispensable AA utilization and metabolism in the pig. In summary, there are no differences in true ileal CP and AA digestibility coefficients in barley samples as determined in this study and the average values reported in the literature. The endogenous CP and AA outputs, determined from this study in the growing-

12 1604 Fan and Sauer finishing pig fed barley-based diets, are considerably higher than previously reported values. The use of digestibility markers, namely, C 2 O 3 and AIA, had dramatic effects on the determination of true ileal CP and AA digestibility coefficients and the endogenous CP and AA outputs in the ileal digesta. It is concluded that dual digestibility markers should be used to measure true ileal CP and AA digestibility coefficients and corresponding endogenous CP and AA outputs when dietary fiber content is high and ileal digesta is collected through a simple-t cannula in the pig. The gastrointestinal endogenous AA secretion, recycling, and losses are important in whole-body AA utilization and homeostasis. Implications True ileal rather than apparent ileal amino acid digestibility coefficients determined in barley samples should be used in diet formulation for swine. 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