C.F.M. de Langel, W. B. Soufff-an? and W. C. Saue: University of Alberta, Edmonton, Alberta T6G 2P5 and Academy of Agricultural Sciences, Oskar Kellner Institute Rostock, German Democratic Republic ABSTRACT
Twelve gilts, with an average initial BW of 38 kg, were fitted with a simple T-cannula at the distal ileum and two catheters in the external jugular veins. The animals were fed 700 g of one of the experimental diets, twice daily at 0800 and 2000. Diets 1 and 2 were cornstarch-based diets formulated to contain 16% CP from soybean meal and canola meal, respectively. Diets 3 and 4 contained 93.9% wheat and barley, respectively. Chromic oxide was included to determine nutrient digestibilities. Following recovery from surgery, "N-leucine was infused continuously via a jugular catheter for 9 d at a rate of 40 mgkg BW-'.b'. Blood samples were taken at every feedin . Ileal digesta were collected for 24 h on d 7 and 9 of the infusion period. Assuming that the N-enrichment excess in the trichloroacetic acid-soluble fraction of the blood was similar to that in endogenous N, the contribution of endogenous to total N in ileal digesta was calculated. The amount of endogenous protein (N x 6.25) recovered in digesta collected from the distal ileum were 25.5,30.5,27.4 and 27.7 g/kg DM intake for the pigs fed diets 1,2.3 and 4, respectively. In the same order for the diets, the real ileal protein digestibilities were 97.5,84.1,99.0 and 94.2%, respectively. In addition, the real ileal amino acid digestibilities were calculated. The recoveries of endogenous protein in ileal digesta and the real ileal protein and amino acid digestibilitieswere higher than those determined by feeding protein-free diets for determining the recovery of endogenous protein and amino acids in ileal digesta. (Key Words: Pigs, Endogenous Protein, Amino Acids, Ileum, Digesta.)
B
J. Anim Sci. 1990.68:409-418 Introduction
ksley and Knabe, 1984; Sauer and Ozimek, 1986). However, measurements obtained with this method are confounded with endogenous amino acids and therefore are referred to as a p parent digestibilities. Using conventional methods, the endogenous amino acid recoveries cannot be quantified when protein-containing 'Financial support was provided by the Farming for the Future Program of the Alberta Agric. Res. Council and diets are fed (e.g., Carlson and Bayley, 1970). the Nat Sci. and Eng. Res. Council of Canada The authors However, using the "N-isotope dilution techacknowledge the assistance of Irma Schutte and Renee nique, nondigested dietary protein can be differPolzhien. entiated from endogenous protein from the 'Present address: Ralston P u r i Canada ~ Inc., Wooddigestive tract (Souffrant etal., 1981,1986). A p stock, Ontario. 3Forschungszen~mher Tierprcduktion, Durnmr- parent protein digestibilities corrected for the recovery of endo enous protein, determined in stmf-Ratmk, G.D.R. 4Dept. of Anim. Sci., UNv. of Alberta, Edmonton, studies with the * N-isotopedilution technique, Alberta, T6G 2P.5 Canada; to whom requests for reprints are referred to as real digestibilities (Krawielitzki should be addressed. et al,, 1977). This technique allows simultaneous Received December 21, 1988. study of factors that affect the real protein digestAccepted June 26,1989. The ileal analysis method is the preferred method for the determination of amino acid digestibilities in feedstuffs for pigs at present (Tan-
f
409
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REAL ILEAL PROTEIN AND AMINO ACID DlGESTlBlLlTlES IN FEEDSTUFFS FOR GROWING PIGS AS DETERMINED WITH THE '5N-ISOTOPE DILUTION TECHNlQUE'
DE LANGE ET A L
410
TABLE 1. FORMULATION OF THEEXPERIMENTALDIETS soybean meal
Ingredients, Q
meal
Wheal
Barley
35.5 44.5 93.9
Dextrose
Canola oil Iodized salt Calcium carbonate Dicalcium monophosphate Vitamin mixture' Mineral mixture' chromic oxide Total
48.3 10.0 3.0 .5
44.0 10.0 3.0 .5
.8
.7
1.1 .2
.1
.2
.5
.1 .5
100.0
100.0
3.0 .5 1.1
93.9 3.0 .5
.7
1.1 .7
.2 .1 .5
.2 .1 .5
100.0
100.0
T h e vitamin and mineral mixture provided the fdlowing per kilogram of diet: 1,300 IU vitamin A, 150 Tu vitamin D3; 11 IU vitamin E;2 mg vitamin K3; 2.2 mg riboflavin; 12 mg niacin; 11 mg pantothenic acid; 11 Fg vitamin 812; 550 mg choline; 1.1 mg thiamine; 1.1 mg pyridoxine; .1 mg biotin; .6mg fdic acid; 50 mg Fe; 50 mg Zn; 2 mg Mn; 3 mg Cu;.14 mg I; .I5 mg Se.
ibilities and those that influence the endogenous protein recoveries. In addition, the real ileal amino acid digestibilities can be calculated from the real protein digestibilities obtained with the 'SN-isotope dilution technique, if assumptions are ma& concerning the amino acid composition of endogenous protein recovered at the distal ileum (de Lange et al., 1989a,b). Studies were conducted u)determine the real ileal protein and amino acid digestibilities in diets containing wheat, barley, canola meal and soybean meal fed to pigs. Materials and Methods
Gild from the University of Alberta swine herd, with an average initial BW of 38 kg, were fitted surgically with a simple T-cannula at the distal ileum according to procedures adapted from Sauer et al. (1983). The design of the cannulas was modified according to de Lange et al. (1989a). Following surgery, the animals were housed individually in stainless steel metabolic crates in a temperature-controlled barn (20 to 22'C). During recovery from surgery, the pigs were fed increasing amounts of the experimental die& (Table l), until they consumed 700 g twice
'Argyle division of Sherwood Medical, St. Louis, MO. 6Model D Comminutor FritL-mill, Fitzpatrick Company, Elmhurst, IL.
daily at 0800 and 2OOO. Water was freely available from a low-pressuredrinking nipple. At least 7 d following the insertion of the cannulas, two polyvinylchloride catheters' (internal diameter 1.56 mm) were surgically implanted, one into each of the external jugular veins according to procedures described by Weirich et al. (1970). The animals were allowed to recover from the second surgery for at least 3 d, or until they consumed 700 g of the experimental diet twice daily. Four different feedstuffs were included in four experimental diets. Diets 1 and 2 were cornstarch-based, formulated to contain approximately 16% CP (N x 6.25) from soybean meal (SBM) or canola meal (CM), respectively.Dextrose was included to improve the palatability of these diets. Diets 3 and 4 contained 93.9% wheat and barley, respectively. All feedstuffs were ground through a 1 mm mesh screen6.Canola oil was included in the diets to reduce dustiness. Vitamins and minerals were supplemented according to NRC (1979)standards.Chromic oxide (.5%) was included in the diets to determine the nument digestibilities. Following recovery, the animals were fed equal amounts of the experimental diets every hour for 3.5 d. The total daily feed allowance was maintained at 1,400 g/d. A 9-d continuous i.v. infusion of [15N]leucine(95% "N-enrichment), via one of the jugular catheters, was initiated 48
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soybean meal Canola meal Wheat Barley Cornstarch
Canola
REAL ILEAL PROTEIN DIGESTIBLITIES INPIGS
ities in the 12 gilts from which valid observations were obtained. In addition to the determination of the real ileal protein digestibilities, we related rate of whole body protein turnover and N retention to real ileal protein digestibilities with this experimental design. The results of the whole body protein turnover study will be reported separately. Analytical and Statistical Procedures. After the conclusion of the experiment, feces and digesta were pooled per animal and collection day. The pooled samples were weighed, freezedried, weighed once more, ground" through a 1-mm mesh screen, and thoroughly mixed before analyses. Analyses for N and DM content were carried out according to AOAC (1980) procedures. Amino acid analyses were performed following short-time pressure hydrolysis, using an amino acid analyzer12,according to the method described by Kreienbring and Wuensche (1976). The samples were pre-oxidized using performic acid for the analyses of methionine and cysteine (Kreienbring and Wuensche, 1976). Tryptophan was determined with a microbial assay using Lactobacillus plantarium (ATCC 8014) following alkaline hydrolysis (Kreienbring and Wuensche, 1976). Chromic oxide was determined according to Fenton and Fenton (1979). All analyses were performed in duplicate. Prior to chemical analyses of blood plasma samples, the TCA precipitate (prec.2) was resuspended in approximately 10 ml of TCA and centrifuged at 1,OOO x g for 10 min. The supernatant fluid (supern. 3) was added to supern. 2 and considered to be the TCA-soluble fraction of blood and the precursor pool for the synthesis of endogenous protein secretions (Souffrant et al., 1981,1986). Trichloroacetic acid was added to a constant volume, and the pooled supernatant fluid was analyzed for N and 'SN-enrichment. The distillate that remained after N analysis was uantitatively transferred to Kjeldahl bottles for N-enrichment analyses in total N in feces, digesta and the TCA-soluble fraction of the blood. Thereafter, 20 ml of 40% sodium hydrox71srnaticM P 13. Ismatic S.A., Limmastrasse 107/109, ide and zinc chips were added and the ammonia Zurich, Switzerland. was redistilled into a beaker containing 50 ml of 'Ruesch, Federal Republic of Germany. 9Citation 111 R2B, Ingram and Bell, Don M i l l s , On- .01N HCI. The samples were re-distilled to separate the pH indicator, which interfered with the tario, Canada. loSorvallGLC-ZB, Dupont Company,Biomedical Di- emission spectrometer, from the ammonia. The vision, Newton, CT. distillation was completed when 100 ml of fluid "Model 4 Laboratory Wiley Mill, Arthur H. Thomas was distilled into the beakers. The water was Cornpny. Philadelphia, PA. evaporated from the ammonium chloride soluMkrotechna AA 88 1, Mikrotechna,Prague,Czchc+ slavakia. tion in an oven at 60°C. The remaining ammo-
's
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h after the start of hourly feeding. Approximately 40 mg ['5Nlleucine, dissolved in a sterile saline solution, was infused per kilogram BW per day. A perjstaltic pump' was used to infuse the solution at a rate of approximately 575 ml per day. Blood and urine samples were taken every 2 h during the fist 36 h of the infusion period; the pigs were fed hourly to study the rate of whole body protein turnover. Urine was collected via balloon cathete? (sue 18) and a closed urinary drainage system'. Following the 3.5-d hourly feeding period, the pigs were fed twice daily 700 g of the experimental diets, at 0800 and 2000, for a period of 7.5 d. Blood and urine samples were taken every 12 h at feeding time. Throughout the 9-d infusion period, feces and urine were collected quantitatively to determine the fecal and urinary ''N excretion. Ileal digesta were collected continuously for 24 h on d 7 and 9 of the infusion period as described previously by de Lange et al. (1989a). Approximately 20 ml of blood was withdrawn from the catheter that was used for sampling. After sampling, the blood was immediately centrifuged" for 10 min at 1,OOO x g. Two 6-ml samples were taken from the supernatant fluid (supern. 1) from each blood sample. The precip itate (prec. 1) was discarded. Ten percent trichloroacetic acid (TCA) was added to the supernatant fluid (1: 1, v:v) and centrifugedat 1,OOO x g for 10 min. The supematant fluid (supern. 2) and the precipitate (prec. 2) were separated and stored at 4'C until further analyses. The excretion of urine was measured volumetricallyevery 2 or 12 h. The urine was acidified with 5 % HC1 to prevent ammonia losses. It then was subsampled. The samples were stored at 4°Cuntil further analyses. Feces and ileal digesta were frozen immediately upon collection. At the conclusion of the infusion period, the animals were slaughteRd to determine whether cannulation had caused intestinal abnormalities. Cannulationdid not result in intestinal abnormal-
411
412
DE LANGE ET A L
where Ne = endogenousN in digesta,Ndig= total N in digesta. fi = percentage enrichment in feed. E& = percentage enrichment in digesta, and Ep1 = percentage enrichment in the TCA-soluble fraction of the blood. The real ileal protein digestibilities then were calculated from the apparent ileal protein digestibilitiesand the recoveries of endogenousprotein (N x 6.25) in ileal digesta as determined by aid of the "N-isotope dilution technique (Souffrant et al., 1981). The "N-enrichment excess in the endogenous protein secretions were assumed to be similar to the average "N-enrichment excess in the TCA-soluble fraction of the three blood samples that were taken during the collection of digesta. The real ileal amino acid digestibilities, in turn, were calculated from the apparent amino acid digestibilities and the recovery of endogenous amino acids in ileal digesta. In these calculations, the amino acid composition of endogenousprotein in ileal digesta was assumed to be similar to that of endogenousprotein in ileal digesta of pigs fed a protein-free diet and simultaneously administered a well-balanced amino acid mixture parenterally, as reported previously by de Lange et al. (1989b). Amino acid analyses in the relevant samplesof the studiesby de Lange et al. (1989b) were repeated to conform to the same method of analyses that were carried out in the present study (Table 2). Valid observations were obtained from three animals per diet. Because problems with feed intake were initially observed, the start of hourly feeding and ['SN]leucine infusions were delayed in some in-
TABLE 2. AMINO ACID COMPOSITION OF ENDOGENOUS PROTElfl Amino acids
gl16 g N
Indispensable Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine
3.11 1.19 3.35 5.23 4.01 1.28 3.56 6.49 .89 4.68
Dispensable Alanine Aspartic acid
5.15 8.54 2.85 9.88 7.12 5.74 5.27 3.92
Cysteine Glutamic acid Glycine Proline Serine Tyrosine
'According to de Lange et al. (1989b) and re-analyzed amcking to Kreienbring and Wuensche (1976).
stances. As a result, observations were not obtained in three experimental periods with four animals per period. as originally planned, and variation resulting from experimental period, therefore, could not be included in the statistical model. However, in a previous experiment, the experimentalperiod had no effect on the recovery of endogenous protein at the distal ileum (de Lange et al., 1989a). Two-way analyses of variance were used with diet and collection day as sources of variation (Harvey, 1960). Treatment means were compared using the Student Newman-Keuls multiple range test (Steel and Tome, 1980). Apparent protein digestibilities corrected for the recovery of endo enous protein, determined in studies with the 'N-isotope dilution technique, are referred to as real digestibilities (Krawielitzki et al.. 1977). Apparent protein digestibilities corrected for the recovery of endogenous protein, determined with conventional methods, are referred to as true digestibilities.
5
Results and Discussion
The proximately analyses and amino acid composition of the four diets are shown in Table '31soniwrnatRFT5201, VEB Statron,Fuerstenwalde. 3. The amino acid composition of the proteincontaining ingredients was similar to that reG.D.R.
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nium chloride was solubilized in 20 ml distilled water. This solution was introducedinto an emission spectr~meter'~ for "N-enrichment analysis. Ileal nutrient digestibilities were determined twice in each pig, using the 24 h pooled digesta samples, collected on d 7 and 9 of the infusion period, respectively. From the ratio "N-enrichment in total N in ileal digesta (including NPN and urea) and the "N-enrichment in total N in the TCA-soluble fraction of the blood, the contribution of endogenous to total N in ileal digesta can be calculated according to the followingformula:
413
REAL ILEAL PROTEIN DIGESTIBILTIllES IN PIGS
TABLE 3. PROXIMATE ANALYSES AND AMINO ACID CONTENT OF THE EXPERIMENTAL DIETS Canola
Item
Crude protein Ether extract Crude fiber Nitrogen-free extract Amino acids, g/16 g N Indispensable Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Dispensable Alanine Aspartic acid Cysteine Glutamic acid Glycine proline
Serine Tyrosine
meal
Wheat
Barley
89.6 18.6 4.1 1.5 70.7
90.5 16.9 5.1 6.2 67.1
87.3 14.4 5.3 2.6 73.4
88.2 11.2 5.3 3.7 75.2
6.77 2.33 4.74 7.97 5.94 1.47 4.83 3.44 1.27 4.29
5.62 2.3 1 4.46 7.69 5.58 1.92 4.13 4.25 1.16 4.93
4.54 2.02 3.99 7.30 2.52 1.67 4.70 2.37 1.17 4.20
4.58 1.85 4.15 7.68 3.55 1.63 5.20 3.37 .99 4.95
4.30 11.40 1.67 18.62 4.26 4.86 5.64 2.97
4.54 7.80 2.48 17.91 5.28 6.40 4.93 2.54
3.61 5.11 2.48 30.69 4.18 10.26 5.11 2.45
4.09 6.00 2.54 23.93 4.03 11.81 4.35 2.7 1
'Dry matter basis.
ported in NRC (1988). The apparent protein and amino acid digestibilities (Table 4) usually were within the range of previously published ileal digestibilities in these feedstuffs (Sauer and Ozimek, 1986). The apparent protein and amino acid digestibilities in the CM diet were lower (P < .05) than in the SBM diet, except for proline. The differences in apparent digestibilities were 17.8,16.3,7.8,15.5 and 20.4 percentage unitsfor protein, lysine, methionine, threonineand tryptophan, respectively, The apparent digestibilities for protein and all the amino acids, with the exception of lysine and threonine, were lower (P < .OS) in barley than in wheat. The difference in apparent protein digestibility between wheat and barley was 10.5 percentage units. Apparent protein digestibilities are affected by the amount of endogenous protein recovered at the distal ileum. When true protein digestibilities are calculated in a conventional manner, it is assumed that the endogenous protein recoveries did not differ when different protein-containing
diets were fed and that these recoveries can be estimated by feeding a protein-free diet (de Lange et al., 1989a,b). If the endogenous protein recoveries are assumed to be 12.7 g/kg DM inrake, as was determined in a study in which pigs were fed a protein-free diet and simultaneously administered amino acids i.v. (de Lange et al., 1989b), the indirectly calculated true protein digestibilities were 90.7,73.5,88.8 and 80.8% for the SBM, CM, wheat and barley diets, respectively (Table 5). Similarly, the true amino acid digestibilities were calculated. Adifferentiation can be made between endogenousprotein secretionsand non-digesteddietary protein in ileal digesta or feces of pigs fed a protein-containingdiet when the "N-isotope dilution technique is used (Souffrant et al., 1981, 1986). The real protein digestibilities in feedstuffs thus can be estimated directly. With this technique, endogenous rather than dietary protein is labeled. If labeled amino acids are administered orally, they will be absorbed and will
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Proximateanalyses, %" Dry-
meal
414
DE LANCE ET A L
TABLE 4. APPARENTILEAL PROTEIN, DRY MAlTER AND AMINO ACID DIGESTIBILITIES (%) IN THE EXPERIMENTAL DIETS
canola meal
meal
Wheat
Barley
SE
Protein Dry matter
83.8' 81.7'
66.0b 66.4d
80.01 75.2b
69Sb 70.4'
1.6 .6
91.9. 87.7" 85.7" 85.6a 87.3' 87.6' 82.P 75.2. 83.5. 81.9.
80.3h 77.4= 69.4' 72.3' 71.0b 79.8b 68.4' 59.7b 63.1b 67.P
84.0b 83.4b 83.5. 71.4b 87.6' 85.9. 66.0b 79.9. 81.7'
76.5' 74.8' 75.ob 77.9 69.gb 78.ob 75.6b 65.6b 59.2b 76.7b
1.7 1.0 1.0 .9 1.8 1.2 1.8 1.9 3.0 1.0
81.4. 85.5. 74.9 87.2b 74.4' 80.2* 87.4. 86.3a
71.gb 65.6' 62.8' 81.1' 60.7b 61.7ab 73.ob 71.9'
75.9 74.4b 8 1.9' 94.2' 61.gb 73.0. 85.4. 86.0.
65.3' 67.3' 74.7b 87.1b 43.2' 48.ob 75. Ib 79.1b
1.5 1.4 1.3 .7 3.5 6.9 1.3 1.4
Amino acids Indispensable Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Dispensable Alanine Aspartic acid Cysteine Glutamic acid Glycine proline
Serine Tyrosine
85.1'
.*bk*dValues in the same row followed by different superscript letters differ (P c .Os).
reappear in the gasuo-intestinal tract via pancreatic secretions within 6 h (Simon et al., 1983), complicating the differentiation between endogenous protein and nondigested dietary protein in the digestive tract. A basic requirement of the "N-isotope dilution technique is the establishment of steady-state conditions, which can be achieved via a continuous i.v. infusion of ["NIleucine. Due to transamination,the "N-labd also will appear in some of the other amino acids, so that not only the N in leucine is labeled (Matthews et al., 1979). The time course in the "Nenrichment excess in the several N - p l s showed that, after an initial period of rapid rise, the "Nenrichment excess in the different N-pools tended to plateau (Figure 1). During the last few days of the infusion period the "NeMchment excess still rose slowly due to the recycling of labeled amino acids (Waterlow et al., 1978). In addition to infused "N, 15N originating from degraded body proteins will contribute to the "N-enrichment excess. For calculating the contribution of endogenous to total protein in ileal digesta, the TCA-soluble fraction of blood was considered to be the precursor pool for the syn-
thesis of endogenous protein secreted into the gastreintestinal lumen (Souffrant et al., 1981, 1986). The TCA-soluble fraction of blood contains free amino acids that are used for the synthesis of endogenous protein. Alpers (1972) demonstrated that amino acids, absorbed from the gastro-intestinal lumen, will also be used for protein synthesis in the intestinal wall without
Time (h) Figure 1. 'lime course of "N-enrichment excess in wine, digesta and the trichloroaatic acid (TCA)-soluble fraction of blood in a pig f,? a soybean meal diet and continuously administered [ Nlleucine i.v.
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Item
415
REAL ILEAL P R O m DIGESTIBILITIES IN PIGS TABLE 5. TRUE ILEAL PROTEIN AND AMINO ACID DIGESTIBILITIESa(%) IN THE EXPERIMENTALDIETS
meal
CanOla meal
Protein
90.7b
73.5d
95.1b 91.1b 90.6b
Amino acids Indispensable Arginine Histidine Isoleucine Leucine
Lysine Methionine Phenylalanine Threonine Tryptophan Valine Dispensable Alanine Aspartic acid Cysteine Glutamic acid Glycine Proline Serine Tyrosine
go.@ 92.e 93 .Sb 87.1' 88.@ 88.3b 89.3bc 89Sb 90.Sb 86.2c
903 S5.Sb 88.3b 93.8b 95.3'
Barley
SE
88.8b
80.8'
1.6
84.4d 81.3' 75.od 77.4d 76.4d 84.8' 74.9d 71.1' 68.8' 74.ld
90.1'
84.Zd
1.7
S8.Sb
82.1'
90.9b 91.4b 85.4' 94.4b 92.6b
84.1' 85.2'
1.0 1.0
80.3' 73Ad 71.4d 85.2d 70.8*
88.1b
68.4b
81.1d 83 .4d
Wheat
90.2b
tE1.6~ 91.6b 89.1b 92.ob 97.ob 76.9bc 77.9b 94.6b 100.lb
82.6'
.9 1.8
87.V
1.2
83.4' 87Sb 69.4' 87.4'
1.8 1.9 3.0 1.0
79.6' 83.5' 87.5' 91.8' 63.2d 53.5'
1.5 1.4 1.3 .7 3.5 6.9 1.3 1.4
88.8'
95.5'
'Indirectly calculated, assuming 12.7 g endogenous protein per kilogram of DM intake and the amino acid composition of endogenous protein to be similar to that observed in pigs fed a protein-free diet and simultaneously administered with amino acids parentally (de Lange et al., 1989b). b3c,dValues in the s a r m row followed by different superscript letters differ (P< .OS).
having to enter the main blood circulation. This implies that the 'SNenrichmentexcess in amino acids, which argactually used for the synthesisof endogenous protein secretions, is lower than the excess measured in the TCA-soluble fraction of the blood. As a result, the contribution of endogenous to total protein in the gastro-intestinaltract is underestimated. Urea-N present in the TCAsoluble fraction of the blood also contributes to the "N-enrichment excess in the TCA soluble fraction. The "N-enrichment excess in urinary N usually was higher than that in the TCA-soluble fraction of blood (Figure 1). This implies that the "N-enrichment excess in urea was probably higher than in the free amino acids in the blood, which probably also resulted in an underestimation of the actual contribution of endogenous to total protein in the gastro-intestinal tract. The "N-enrichment excess in digesta, expressed as a percentage of the excess in the TCAsoluble fraction of blood, shows the relative contribution of endogenous to total protein in ileal digesta. These relative contributionsdid not
differ between d 7 and d 9 of the infusion period (P > .lo). Although no plateau was reached in the "N-enrichment excess in the different N pools, no differencewas observed in the relative contribution of endogenous protein to total protein in ileal digesta between d 7 and d 9 of the infusion periods. The real protein digestibilities therefore were calculated from these results. The amount of endogenous protein in ileal digesta varied between 25.5 and 30.5 g/kg DM intake (Table 6). These values were much higher than those observed in a previous experiment in which pigs were fed a protein-free diet while amino acids were simultaneously administered i.v. (de Lange et al., 1989) or the values observed in studies in which pigs were fed a protein-free diet (Wuensche et al., 1987; de Lange et al., 1989a). The quantities observed in the present study also were larger than those in studies in which alternative methods were used to determine the recovery of endogenous protein at the distal ileum in pigs: approximately 17.6 and 19.3 g per kg DM intake in pigs fed a barley-soybean
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Item
4 16
DE LANGE ET A L
TABLE 6. ENDOGENOUS PROTEIN IN ILEAL YJGESTAOF PIGS FED PROTEIN-CONTAINING DIETS AS DETERMINED WITH THE N-ISOTOPEDILUTION TECHNIQUE Item ~
Endogenous protein g per kg DM intake 96 of Total CP g per 100 g CPintake
Canda meal
m e a t
Barley
SE
25.5
30.5 53.9 18.0b
27.4 94.5. 19.1b
27.7 81.1b 24.7'
2.4 3.0 1.7
~~
84.6b
13.7b
8*b*c*dValua in the same row followed by different superscript letters differ (P < .OS).
meal diet (Zebrowska et al., 1982) and a fish meal-wheat diet (Simon et al., 1987), respectively. Zebrowska et al. (1982) calculated the contribution of endogenous to total protein in ileal digesta from the amino acid composition of dietary, endogenous and total protein in ileal digesta assuming that the amino acid composition of endogenous protein was constant and that the true or real digestibilities were similar for all amino acids. The last assumption, in particular, can be questioned because feedstuffs contain a variety of different proteins. The amino acid composition and the real ileal digestibilities of these proteins may vary. Simon et al. (1987) used an alternative 15~-isotope dilution technique in which proteincontaining diets were fed to pigs that were previously labeled with "N by feeding ["~~ammonium carbonate and lam ammonium chloride and in which the "N-enrichment excess in endogenousprotein was assumed to be similar to that in urinary N. Because the "N-enrichment excess in urinary N was higher than that in the TCA-soluble fraction of the blood in the resent study, it can be questioned whether the %-enrichment excess in urinary N is a valid indicator for that in endogenous protein. Only when the microbes present in the gastro-intestinal tract preferentially incorporate N originating from urea into microbial protein and when the conhibution of microbial N to total N in ileal digesta is larger than was previously estimated (Dierick et al., 1986), would the "Nenrichment excess in urine provide a better estimate of the enrichment excess in endogenous protein than that in the TCA-soluble fraction of the blood. In addition, differences in diet composition may contribute to the differences in the recovery of endogenous protein at the distal ileum in the present study and in studies by Zebrowska et al. (1982) and Simon et al. (1987). The present study showed that the indirectly
P
calculated true protein digestibilities underestimate the real protein digestibilities (Figure 2). The relatively low real protein digestibility in the CM diet (84.1%, Table 7) is probably related to its high fiber content (Bell, 1984). Components such as lignin, pectins, tannins and cellulose in CM may interfere with the digestion of protein in this feedstuff (Bell, 1984). Furthermore, the real protein digestibility in the barley diet was lower than 100% (94.296,Table 7). Eggum and Christensen (1975) showed that tannins, which are present in barley, may bind to protein (amino acids) and thereby affect the digestion and (re-) absorption of both endogenous and exogenous protein. Krawielitzki et al. (1977), in studies with rats, showed a linear relationship between the amount of dietary protein and the excretion of endogenous protein in feces. suggesting that the recovery of endogenous protein should be presented in units other than grams/kilogram DM intake, especially if the protein contents of the diets vary. Differences still were apparent (P c .05) between
EXlApparent
O T r u e (conv)
m R e a l ('5N)
Figure 2 Apparent, hue and real ileal protein digestibilities in the experimentaldiets.
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~~
Soybean meal
417
REAL ILEAL PROTEIN DIGESTIBILITIES IN PIGS TABLE 7. REAL ILEAL PROTEIN AND AMINO ACID DIGESTIBILITIEe (40) IN THE EXPERIMENTALDIETS
Item
Amino acids Indispensable Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Dispensable Alanine Aspartic acid Cysteine Glutamic acid Glycine proline Serine Tyrwine
Canola meal
Wheat
Barley
SE
97.9
84.ld
99.e
94.2'
1.0
98.Zb 94.6b 95.4c 949
90.2'
97.1b 94.7b
93.3c 90.7' 95.v 94.3c 97.7' 97.9
1.1 .9 1.2 1.1 1.2 1.2 1.8 3.3 3.0 1.7
96.6' 999 9 2 1' 101.w 93.1b %.gC
97.V 95.7' 97.9b 94.9 97.3b %.4b 100.2' 104.4'
86.7d 82.9d 84.9 M.od 91.8d 83.9d 87.2d 76.9' 84.ld 92.3* 85.3d 83.5'
91.v
85.P 77.9b 92.3d 99.7'
99.9 98Ab I01 .7b 102.2b 100.3b 118.2b W.4b 1O3.e l02.P l06.2b 103.Sb 100.3b 94.3b 83.7b 105.lb l16Sb
92.5' 113.3b 81.5' 100.lbc
96.9
1.4
1 02.6b 102.5b 97.4c 86.9c
1.8 21 .8 24 6.3 1.3 2.4
6o.v
104.9b 114.9b
'Directly determined real protein digestibilities using the "N-isotope dilution technique. The real amino acid digestibilities are estimated from the apparentprotein and amino acid digestibilities. the real protein digestibilities and the amino acid composition of endogenous protein as observed in pigs fed a protein-free diet and simultaneously administered with amino acids parentally (de Lange et al.. 1989b). b~c*d.eValues in the same row followed by different superscript letters differ (Pc .Os>.
diets if the recovery of endogenous protein was expressed as grams/l00 g CP intake (Table 6). The recovery was highest for the barley (24.7 g/lOO g CP intake) and lowest for the SBM diet (13.7g/lOogCPintake). Intheory,theseresults suggest that, if the four feedstuffs would have been compared at a similarprotein, barley would have resulted in the largest endogenous protein recoveries. More studies are necessary to identify the factors in these feedstuffs that are responsible for the observed differences in endogenous p r e tein recoveries. The aforementioned considerations also illustrate that difficulties may arise in the interpretation of apparent protein digestibilities, especially when the protein contents of the experimental diets vary and when different p r e tein sources are included in one diet (Imbeah et al., 1988). The N-isotope dilution technique only measures the recovery of total endogenous protein in ileal digesta or feces, not the recovery of the individual endogenous amino acids. In order to
calculate the real ileal amino digestibilities, the amino acid composition of endogenous protein was assumed to be similar to the amino acid composition of endogenous protein observed in a previous experiment in which a protein-fiee diet was fed to pigs while amino acids were administeredsimultaneously i.v. (de Lange et al., 1989b, Table 7). For some of the amino acids, especially in the wheat diet, the calculated real amino acid digestibilities exceeded 100%.Because the content of some of the amino acids in wheat was very low (Table 3) and the real protein digestibility was extremely high (Table 7), a small overestimation in the content of these amino acids in endogenous protein may result in overestimationof md amino acid digestibilities. Measuring the "N-enrichment excess in the individual amino acids as opposed to N in blood plasma and digesta may provide a better estimate of the real amino acid digestibilities. These results, however, indicate that the real amino acid digestibilitiesare higher than the true digestibil-
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Prorein
SOY-
meal
418
DE LANCE ET AL.
ities determined in conventional digestibility studies (Tables 5 and 7). Implications
Literature Cited
Alpers, D. H. 1972.Protein synthesis in intestinal mucosa: the effect of route of administration of precursor amino acids. J. of Clin. Invest 51:167. AOAC. 1980. Official methods of analyses (12th Ed.). Association of Official Analytical Chemists, Washington, DC. Bell, J. M. 1984. Nutrients and toxicants in rapeseed meal: A review. J. Anim Sci. 5 8 9 6 . Carlson, K. H. and H. S. Bayley. 1970. Nitrogenandamino acids in the feces of young pigs receiving a p t e i n free diet and diets containing graded levels of soybean meal or casein. 1. Nutr. 100:1353. de Lange, C.F.M., W. C. Sauer, R. Mosenthin and W. B. Souffrant. 1989a. The effwt of feeding different protein-free diets on the recovery and amino acid composition of endogenous protein collected from the distal ileum and feces in pigs. J. Anim. Sci. 67:746. de Lange, C.F.M.. W. C. Sauer and W. B. Souffrant. 1989b. The effect of protein status of the pig on the recovery and amino acid composition of endogenous protein in digesta collected from the distal ileum. J. Anim Sci. 67:755. Dierick, N. A., I. J. Vervaeke, J. A. Decuypere and H. K. Henderickx. 1986. Influence of the gut flora and of some growth-promoting feed additives on nitrogen metabolism in pigs. 11. Studies in vivo. Livest. prod. Sci. 14177. Eggum, B. 0. and K. D. Christenson. 1975. Influence of tannin on protein utilization of feedstuffs with special reference to barley. In: Breeding for Seed Im provement Using Nuclear Techniques. p 135. International Atomic Energy Agency, Vienna Fenton, T.W. and M. Fenton. 1979. An improved procedure for determination of chromic oxide in feed and feces. Can. J. Anim Sci. 59:631. Harvey, W. R. 1960. Least squan: analyses of data with unequal subclass numbers. USDA publ. ARS 2@8, Washington, DC. Imbeah, M.,W. C. Sauer and R. Mosenthin. 1988. The prediction of the digestible amino acid supply in
York. Tanksley, E D. and D. A. Knabe. 1984. Ileal digestibilities of amino acids in pig feeds and their use in formulating diets. In: W. Haresign and D.J.A. Cole (Ed.) Recent Advances in Animal Nutrition. p 75. Butterwarths, London. Waterlow. J. C., P. J. Garlick and D. J. Millward. 1978. Protein tllmover in mammalian tissues and in the whole body. North Holland, Amsterdam. Weirich, W.E., J. A. Will and C. W. Crumpton. 1970. A technique for placing chronic indwelling catheters in swine. J. Appl. Physiol. 28:117. Wuensche, J., U. Hermann, M. Meinl, U. Hennig, E Kreienbring and P. Zwien. 1987. Einfluss exogener Faktoren auf die praezaekaleNaehrstoff und Amine saeurenresorption ermittelt an Schweinen mit IleoRektal-Anastomosen. Arch. Tieremaehr. 37:745. Zebrowska, T., 0. Simon, R. Muenchmeyer, E. Wolf, €1. Bergner and H. Zebrowska 1982. Flow of endogenous and exogenous amino acids along the gut of pigs. Arch. Tierernaehr. 32431.
Downloaded from https://academic.oup.com/jas/article-abstract/68/2/409/4703995 by Iowa State University user on 17 January 2019
The 15N-isotopedilution technique was used to determine the recovery of endogenous protein at the distal ileum in pigs. Endogenous protein was much higher than previously determined in studies in which protein-free diets were fed. The differences in apparent protein (and amino acid) digestibilities between the feedstuffs seemed U, be affected more by differences in the endogenous protein (and amino acid) recoveries than by differences in real protein (and amino acid) digestibilities. Studies on factors that affect the recovery of endogenous protein in ileal digesta and real amino acid digestibilitiesusing the "Nisotope dilution techniquedeserve more attention in future research.
barley-soybean meal ox canola meal diets and pan=tic secretions in pigs. J. Anim Sci. 66:1409. Kmwielitzki, K., T. Volker, S. Smulikowska, H. D. Bock and J. Wuensche. 1977. Weitex Untersuchungen zum Multikomptment-Modell des Protein-Stoffwechsels. Arch. Tieremaehr. 27:609. Kreienbring, E and I. Wuensche. 1976. Darlegung anwendbare Methoden zur Proteinhydrolyse und Aminosauerenbestimmung. Tag. Ber. Akad. Landwirtsch. Wiss. GDR (Berlin) 142:27. Matthews, D. E., E. B. Ben-Galimand D. M. Bier. 1979. Determination of stable isotopic enrichment in individual plasma amino acids by chemical ionizationmass spectrometry. Anal. Chem 51:80. NRC. 1979. Nutrient Requirements of Swine. (8th Rev. Ed.) National Academy Press, Washington, DC. NRC. 1988. Nuuient Requirements of Swine. (9th Rev. Ed.) National Academy Press, Washington, DC. Sauer, W. C., H. Jorgensen and R. Benins. 1983. A modified nylon bag technique for determining apparent digestibilities of protein in feedstuffs for pigs. Can. J. Anim Sci. 63233. Sauer, W.C. and L. Ozimek. 1986. Digestibility of amino acids in swine: results and theirpractical application. A =view. Livest. Prod. Sci. 15367. Simon, O., H. Bergner and I. G. Partridge. 1987. Estimation of endo enous N proportions in ileal digesta and faeces in kN-labelled pigs. Arch. Tierernaehr. 10851. Simon, O., T. Zebrowska, H. Bergner and R. Muenchmeyer. 1983. Investigations on the pancreatic and stomach secretions in pigs by means of continuous infusion of 14Camino acids. Arch. Tiexrnaehr. 33:9. Souffrant,W. B.. B. Darcy-Vrillon, T. Coning, J. P. Laplace, R. Koehler, G. Gebhardt and A. Rerat. 1986. Recycling of endogenous nitrogen in the pig. Arch. Tieremaehr. 36269. Souffrant, W. B., R. Koehler, R. Matkowitz, G. Gebhardt and H. Schmandke. 1981. Ernaehrungsphysie logische Untersuchungen an Schweinen zur beurteilung von modifierten Roteinen. Arch. Tieremaehr. 3 1675. Steel, R.G.D. and J. H. Tome. 1980. Principles and Prp cedures of Statistics. McGraw-Hill Book Co., New
Twelve gilts, with an average initial BW of 38 kg, were fitted with a simple T-cannula at the distal ileum and two catheters in the external jugular veins. The animals were fed 700 g of one of the experimental diets, twice daily at 0800 and 2000. Diets 1 and 2 were cornstarch-based diets formulated to contain 16% CP from soybean meal and canola meal, respectively. Diets 3 and 4 contained 93.9% wheat and barley, respectively. Chromic oxide was included to determine nutrient digestibilities. Following recovery from surgery, "N-leucine was infused continuously via a jugular catheter for 9 d at a rate of 40 mgkg BW-'.b'. Blood samples were taken at every feedin . Ileal digesta were collected for 24 h on d 7 and 9 of the infusion period. Assuming that the N-enrichment excess in the trichloroacetic acid-soluble fraction of the blood was similar to that in endogenous N, the contribution of endogenous to total N in ileal digesta was calculated. The amount of endogenous protein (N x 6.25) recovered in digesta collected from the distal ileum were 25.5,30.5,27.4 and 27.7 g/kg DM intake for the pigs fed diets 1,2.3 and 4, respectively. In the same order for the diets, the real ileal protein digestibilities were 97.5,84.1,99.0 and 94.2%, respectively. In addition, the real ileal amino acid digestibilities were calculated. The recoveries of endogenous protein in ileal digesta and the real ileal protein and amino acid digestibilitieswere higher than those determined by feeding protein-free diets for determining the recovery of endogenous protein and amino acids in ileal digesta. (Key Words: Pigs, Endogenous Protein, Amino Acids, Ileum, Digesta.)
B
J. Anim Sci. 1990.68:409-418 Introduction
ksley and Knabe, 1984; Sauer and Ozimek, 1986). However, measurements obtained with this method are confounded with endogenous amino acids and therefore are referred to as a p parent digestibilities. Using conventional methods, the endogenous amino acid recoveries cannot be quantified when protein-containing 'Financial support was provided by the Farming for the Future Program of the Alberta Agric. Res. Council and diets are fed (e.g., Carlson and Bayley, 1970). the Nat Sci. and Eng. Res. Council of Canada The authors However, using the "N-isotope dilution techacknowledge the assistance of Irma Schutte and Renee nique, nondigested dietary protein can be differPolzhien. entiated from endogenous protein from the 'Present address: Ralston P u r i Canada ~ Inc., Wooddigestive tract (Souffrant etal., 1981,1986). A p stock, Ontario. 3Forschungszen~mher Tierprcduktion, Durnmr- parent protein digestibilities corrected for the recovery of endo enous protein, determined in stmf-Ratmk, G.D.R. 4Dept. of Anim. Sci., UNv. of Alberta, Edmonton, studies with the * N-isotopedilution technique, Alberta, T6G 2P.5 Canada; to whom requests for reprints are referred to as real digestibilities (Krawielitzki should be addressed. et al,, 1977). This technique allows simultaneous Received December 21, 1988. study of factors that affect the real protein digestAccepted June 26,1989. The ileal analysis method is the preferred method for the determination of amino acid digestibilities in feedstuffs for pigs at present (Tan-
f
409
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REAL ILEAL PROTEIN AND AMINO ACID DlGESTlBlLlTlES IN FEEDSTUFFS FOR GROWING PIGS AS DETERMINED WITH THE '5N-ISOTOPE DILUTION TECHNlQUE'
DE LANGE ET A L
410
TABLE 1. FORMULATION OF THEEXPERIMENTALDIETS soybean meal
Ingredients, Q
meal
Wheal
Barley
35.5 44.5 93.9
Dextrose
Canola oil Iodized salt Calcium carbonate Dicalcium monophosphate Vitamin mixture' Mineral mixture' chromic oxide Total
48.3 10.0 3.0 .5
44.0 10.0 3.0 .5
.8
.7
1.1 .2
.1
.2
.5
.1 .5
100.0
100.0
3.0 .5 1.1
93.9 3.0 .5
.7
1.1 .7
.2 .1 .5
.2 .1 .5
100.0
100.0
T h e vitamin and mineral mixture provided the fdlowing per kilogram of diet: 1,300 IU vitamin A, 150 Tu vitamin D3; 11 IU vitamin E;2 mg vitamin K3; 2.2 mg riboflavin; 12 mg niacin; 11 mg pantothenic acid; 11 Fg vitamin 812; 550 mg choline; 1.1 mg thiamine; 1.1 mg pyridoxine; .1 mg biotin; .6mg fdic acid; 50 mg Fe; 50 mg Zn; 2 mg Mn; 3 mg Cu;.14 mg I; .I5 mg Se.
ibilities and those that influence the endogenous protein recoveries. In addition, the real ileal amino acid digestibilities can be calculated from the real protein digestibilities obtained with the 'SN-isotope dilution technique, if assumptions are ma& concerning the amino acid composition of endogenous protein recovered at the distal ileum (de Lange et al., 1989a,b). Studies were conducted u)determine the real ileal protein and amino acid digestibilities in diets containing wheat, barley, canola meal and soybean meal fed to pigs. Materials and Methods
Gild from the University of Alberta swine herd, with an average initial BW of 38 kg, were fitted surgically with a simple T-cannula at the distal ileum according to procedures adapted from Sauer et al. (1983). The design of the cannulas was modified according to de Lange et al. (1989a). Following surgery, the animals were housed individually in stainless steel metabolic crates in a temperature-controlled barn (20 to 22'C). During recovery from surgery, the pigs were fed increasing amounts of the experimental die& (Table l), until they consumed 700 g twice
'Argyle division of Sherwood Medical, St. Louis, MO. 6Model D Comminutor FritL-mill, Fitzpatrick Company, Elmhurst, IL.
daily at 0800 and 2OOO. Water was freely available from a low-pressuredrinking nipple. At least 7 d following the insertion of the cannulas, two polyvinylchloride catheters' (internal diameter 1.56 mm) were surgically implanted, one into each of the external jugular veins according to procedures described by Weirich et al. (1970). The animals were allowed to recover from the second surgery for at least 3 d, or until they consumed 700 g of the experimental diet twice daily. Four different feedstuffs were included in four experimental diets. Diets 1 and 2 were cornstarch-based, formulated to contain approximately 16% CP (N x 6.25) from soybean meal (SBM) or canola meal (CM), respectively.Dextrose was included to improve the palatability of these diets. Diets 3 and 4 contained 93.9% wheat and barley, respectively. All feedstuffs were ground through a 1 mm mesh screen6.Canola oil was included in the diets to reduce dustiness. Vitamins and minerals were supplemented according to NRC (1979)standards.Chromic oxide (.5%) was included in the diets to determine the nument digestibilities. Following recovery, the animals were fed equal amounts of the experimental diets every hour for 3.5 d. The total daily feed allowance was maintained at 1,400 g/d. A 9-d continuous i.v. infusion of [15N]leucine(95% "N-enrichment), via one of the jugular catheters, was initiated 48
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soybean meal Canola meal Wheat Barley Cornstarch
Canola
REAL ILEAL PROTEIN DIGESTIBLITIES INPIGS
ities in the 12 gilts from which valid observations were obtained. In addition to the determination of the real ileal protein digestibilities, we related rate of whole body protein turnover and N retention to real ileal protein digestibilities with this experimental design. The results of the whole body protein turnover study will be reported separately. Analytical and Statistical Procedures. After the conclusion of the experiment, feces and digesta were pooled per animal and collection day. The pooled samples were weighed, freezedried, weighed once more, ground" through a 1-mm mesh screen, and thoroughly mixed before analyses. Analyses for N and DM content were carried out according to AOAC (1980) procedures. Amino acid analyses were performed following short-time pressure hydrolysis, using an amino acid analyzer12,according to the method described by Kreienbring and Wuensche (1976). The samples were pre-oxidized using performic acid for the analyses of methionine and cysteine (Kreienbring and Wuensche, 1976). Tryptophan was determined with a microbial assay using Lactobacillus plantarium (ATCC 8014) following alkaline hydrolysis (Kreienbring and Wuensche, 1976). Chromic oxide was determined according to Fenton and Fenton (1979). All analyses were performed in duplicate. Prior to chemical analyses of blood plasma samples, the TCA precipitate (prec.2) was resuspended in approximately 10 ml of TCA and centrifuged at 1,OOO x g for 10 min. The supernatant fluid (supern. 3) was added to supern. 2 and considered to be the TCA-soluble fraction of blood and the precursor pool for the synthesis of endogenous protein secretions (Souffrant et al., 1981,1986). Trichloroacetic acid was added to a constant volume, and the pooled supernatant fluid was analyzed for N and 'SN-enrichment. The distillate that remained after N analysis was uantitatively transferred to Kjeldahl bottles for N-enrichment analyses in total N in feces, digesta and the TCA-soluble fraction of the blood. Thereafter, 20 ml of 40% sodium hydrox71srnaticM P 13. Ismatic S.A., Limmastrasse 107/109, ide and zinc chips were added and the ammonia Zurich, Switzerland. was redistilled into a beaker containing 50 ml of 'Ruesch, Federal Republic of Germany. 9Citation 111 R2B, Ingram and Bell, Don M i l l s , On- .01N HCI. The samples were re-distilled to separate the pH indicator, which interfered with the tario, Canada. loSorvallGLC-ZB, Dupont Company,Biomedical Di- emission spectrometer, from the ammonia. The vision, Newton, CT. distillation was completed when 100 ml of fluid "Model 4 Laboratory Wiley Mill, Arthur H. Thomas was distilled into the beakers. The water was Cornpny. Philadelphia, PA. evaporated from the ammonium chloride soluMkrotechna AA 88 1, Mikrotechna,Prague,Czchc+ slavakia. tion in an oven at 60°C. The remaining ammo-
's
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h after the start of hourly feeding. Approximately 40 mg ['5Nlleucine, dissolved in a sterile saline solution, was infused per kilogram BW per day. A perjstaltic pump' was used to infuse the solution at a rate of approximately 575 ml per day. Blood and urine samples were taken every 2 h during the fist 36 h of the infusion period; the pigs were fed hourly to study the rate of whole body protein turnover. Urine was collected via balloon cathete? (sue 18) and a closed urinary drainage system'. Following the 3.5-d hourly feeding period, the pigs were fed twice daily 700 g of the experimental diets, at 0800 and 2000, for a period of 7.5 d. Blood and urine samples were taken every 12 h at feeding time. Throughout the 9-d infusion period, feces and urine were collected quantitatively to determine the fecal and urinary ''N excretion. Ileal digesta were collected continuously for 24 h on d 7 and 9 of the infusion period as described previously by de Lange et al. (1989a). Approximately 20 ml of blood was withdrawn from the catheter that was used for sampling. After sampling, the blood was immediately centrifuged" for 10 min at 1,OOO x g. Two 6-ml samples were taken from the supernatant fluid (supern. 1) from each blood sample. The precip itate (prec. 1) was discarded. Ten percent trichloroacetic acid (TCA) was added to the supernatant fluid (1: 1, v:v) and centrifugedat 1,OOO x g for 10 min. The supematant fluid (supern. 2) and the precipitate (prec. 2) were separated and stored at 4'C until further analyses. The excretion of urine was measured volumetricallyevery 2 or 12 h. The urine was acidified with 5 % HC1 to prevent ammonia losses. It then was subsampled. The samples were stored at 4°Cuntil further analyses. Feces and ileal digesta were frozen immediately upon collection. At the conclusion of the infusion period, the animals were slaughteRd to determine whether cannulation had caused intestinal abnormalities. Cannulationdid not result in intestinal abnormal-
411
412
DE LANGE ET A L
where Ne = endogenousN in digesta,Ndig= total N in digesta. fi = percentage enrichment in feed. E& = percentage enrichment in digesta, and Ep1 = percentage enrichment in the TCA-soluble fraction of the blood. The real ileal protein digestibilities then were calculated from the apparent ileal protein digestibilitiesand the recoveries of endogenousprotein (N x 6.25) in ileal digesta as determined by aid of the "N-isotope dilution technique (Souffrant et al., 1981). The "N-enrichment excess in the endogenous protein secretions were assumed to be similar to the average "N-enrichment excess in the TCA-soluble fraction of the three blood samples that were taken during the collection of digesta. The real ileal amino acid digestibilities, in turn, were calculated from the apparent amino acid digestibilities and the recovery of endogenous amino acids in ileal digesta. In these calculations, the amino acid composition of endogenousprotein in ileal digesta was assumed to be similar to that of endogenousprotein in ileal digesta of pigs fed a protein-free diet and simultaneously administered a well-balanced amino acid mixture parenterally, as reported previously by de Lange et al. (1989b). Amino acid analyses in the relevant samplesof the studiesby de Lange et al. (1989b) were repeated to conform to the same method of analyses that were carried out in the present study (Table 2). Valid observations were obtained from three animals per diet. Because problems with feed intake were initially observed, the start of hourly feeding and ['SN]leucine infusions were delayed in some in-
TABLE 2. AMINO ACID COMPOSITION OF ENDOGENOUS PROTElfl Amino acids
gl16 g N
Indispensable Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine
3.11 1.19 3.35 5.23 4.01 1.28 3.56 6.49 .89 4.68
Dispensable Alanine Aspartic acid
5.15 8.54 2.85 9.88 7.12 5.74 5.27 3.92
Cysteine Glutamic acid Glycine Proline Serine Tyrosine
'According to de Lange et al. (1989b) and re-analyzed amcking to Kreienbring and Wuensche (1976).
stances. As a result, observations were not obtained in three experimental periods with four animals per period. as originally planned, and variation resulting from experimental period, therefore, could not be included in the statistical model. However, in a previous experiment, the experimentalperiod had no effect on the recovery of endogenous protein at the distal ileum (de Lange et al., 1989a). Two-way analyses of variance were used with diet and collection day as sources of variation (Harvey, 1960). Treatment means were compared using the Student Newman-Keuls multiple range test (Steel and Tome, 1980). Apparent protein digestibilities corrected for the recovery of endo enous protein, determined in studies with the 'N-isotope dilution technique, are referred to as real digestibilities (Krawielitzki et al.. 1977). Apparent protein digestibilities corrected for the recovery of endogenous protein, determined with conventional methods, are referred to as true digestibilities.
5
Results and Discussion
The proximately analyses and amino acid composition of the four diets are shown in Table '31soniwrnatRFT5201, VEB Statron,Fuerstenwalde. 3. The amino acid composition of the proteincontaining ingredients was similar to that reG.D.R.
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nium chloride was solubilized in 20 ml distilled water. This solution was introducedinto an emission spectr~meter'~ for "N-enrichment analysis. Ileal nutrient digestibilities were determined twice in each pig, using the 24 h pooled digesta samples, collected on d 7 and 9 of the infusion period, respectively. From the ratio "N-enrichment in total N in ileal digesta (including NPN and urea) and the "N-enrichment in total N in the TCA-soluble fraction of the blood, the contribution of endogenous to total N in ileal digesta can be calculated according to the followingformula:
413
REAL ILEAL PROTEIN DIGESTIBILTIllES IN PIGS
TABLE 3. PROXIMATE ANALYSES AND AMINO ACID CONTENT OF THE EXPERIMENTAL DIETS Canola
Item
Crude protein Ether extract Crude fiber Nitrogen-free extract Amino acids, g/16 g N Indispensable Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Dispensable Alanine Aspartic acid Cysteine Glutamic acid Glycine proline
Serine Tyrosine
meal
Wheat
Barley
89.6 18.6 4.1 1.5 70.7
90.5 16.9 5.1 6.2 67.1
87.3 14.4 5.3 2.6 73.4
88.2 11.2 5.3 3.7 75.2
6.77 2.33 4.74 7.97 5.94 1.47 4.83 3.44 1.27 4.29
5.62 2.3 1 4.46 7.69 5.58 1.92 4.13 4.25 1.16 4.93
4.54 2.02 3.99 7.30 2.52 1.67 4.70 2.37 1.17 4.20
4.58 1.85 4.15 7.68 3.55 1.63 5.20 3.37 .99 4.95
4.30 11.40 1.67 18.62 4.26 4.86 5.64 2.97
4.54 7.80 2.48 17.91 5.28 6.40 4.93 2.54
3.61 5.11 2.48 30.69 4.18 10.26 5.11 2.45
4.09 6.00 2.54 23.93 4.03 11.81 4.35 2.7 1
'Dry matter basis.
ported in NRC (1988). The apparent protein and amino acid digestibilities (Table 4) usually were within the range of previously published ileal digestibilities in these feedstuffs (Sauer and Ozimek, 1986). The apparent protein and amino acid digestibilities in the CM diet were lower (P < .05) than in the SBM diet, except for proline. The differences in apparent digestibilities were 17.8,16.3,7.8,15.5 and 20.4 percentage unitsfor protein, lysine, methionine, threonineand tryptophan, respectively, The apparent digestibilities for protein and all the amino acids, with the exception of lysine and threonine, were lower (P < .OS) in barley than in wheat. The difference in apparent protein digestibility between wheat and barley was 10.5 percentage units. Apparent protein digestibilities are affected by the amount of endogenous protein recovered at the distal ileum. When true protein digestibilities are calculated in a conventional manner, it is assumed that the endogenous protein recoveries did not differ when different protein-containing
diets were fed and that these recoveries can be estimated by feeding a protein-free diet (de Lange et al., 1989a,b). If the endogenous protein recoveries are assumed to be 12.7 g/kg DM inrake, as was determined in a study in which pigs were fed a protein-free diet and simultaneously administered amino acids i.v. (de Lange et al., 1989b), the indirectly calculated true protein digestibilities were 90.7,73.5,88.8 and 80.8% for the SBM, CM, wheat and barley diets, respectively (Table 5). Similarly, the true amino acid digestibilities were calculated. Adifferentiation can be made between endogenousprotein secretionsand non-digesteddietary protein in ileal digesta or feces of pigs fed a protein-containingdiet when the "N-isotope dilution technique is used (Souffrant et al., 1981, 1986). The real protein digestibilities in feedstuffs thus can be estimated directly. With this technique, endogenous rather than dietary protein is labeled. If labeled amino acids are administered orally, they will be absorbed and will
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Proximateanalyses, %" Dry-
meal
414
DE LANCE ET A L
TABLE 4. APPARENTILEAL PROTEIN, DRY MAlTER AND AMINO ACID DIGESTIBILITIES (%) IN THE EXPERIMENTAL DIETS
canola meal
meal
Wheat
Barley
SE
Protein Dry matter
83.8' 81.7'
66.0b 66.4d
80.01 75.2b
69Sb 70.4'
1.6 .6
91.9. 87.7" 85.7" 85.6a 87.3' 87.6' 82.P 75.2. 83.5. 81.9.
80.3h 77.4= 69.4' 72.3' 71.0b 79.8b 68.4' 59.7b 63.1b 67.P
84.0b 83.4b 83.5. 71.4b 87.6' 85.9. 66.0b 79.9. 81.7'
76.5' 74.8' 75.ob 77.9 69.gb 78.ob 75.6b 65.6b 59.2b 76.7b
1.7 1.0 1.0 .9 1.8 1.2 1.8 1.9 3.0 1.0
81.4. 85.5. 74.9 87.2b 74.4' 80.2* 87.4. 86.3a
71.gb 65.6' 62.8' 81.1' 60.7b 61.7ab 73.ob 71.9'
75.9 74.4b 8 1.9' 94.2' 61.gb 73.0. 85.4. 86.0.
65.3' 67.3' 74.7b 87.1b 43.2' 48.ob 75. Ib 79.1b
1.5 1.4 1.3 .7 3.5 6.9 1.3 1.4
Amino acids Indispensable Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Dispensable Alanine Aspartic acid Cysteine Glutamic acid Glycine proline
Serine Tyrosine
85.1'
.*bk*dValues in the same row followed by different superscript letters differ (P c .Os).
reappear in the gasuo-intestinal tract via pancreatic secretions within 6 h (Simon et al., 1983), complicating the differentiation between endogenous protein and nondigested dietary protein in the digestive tract. A basic requirement of the "N-isotope dilution technique is the establishment of steady-state conditions, which can be achieved via a continuous i.v. infusion of ["NIleucine. Due to transamination,the "N-labd also will appear in some of the other amino acids, so that not only the N in leucine is labeled (Matthews et al., 1979). The time course in the "Nenrichment excess in the several N - p l s showed that, after an initial period of rapid rise, the "Nenrichment excess in the different N-pools tended to plateau (Figure 1). During the last few days of the infusion period the "NeMchment excess still rose slowly due to the recycling of labeled amino acids (Waterlow et al., 1978). In addition to infused "N, 15N originating from degraded body proteins will contribute to the "N-enrichment excess. For calculating the contribution of endogenous to total protein in ileal digesta, the TCA-soluble fraction of blood was considered to be the precursor pool for the syn-
thesis of endogenous protein secreted into the gastreintestinal lumen (Souffrant et al., 1981, 1986). The TCA-soluble fraction of blood contains free amino acids that are used for the synthesis of endogenous protein. Alpers (1972) demonstrated that amino acids, absorbed from the gastro-intestinal lumen, will also be used for protein synthesis in the intestinal wall without
Time (h) Figure 1. 'lime course of "N-enrichment excess in wine, digesta and the trichloroaatic acid (TCA)-soluble fraction of blood in a pig f,? a soybean meal diet and continuously administered [ Nlleucine i.v.
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Item
415
REAL ILEAL P R O m DIGESTIBILITIES IN PIGS TABLE 5. TRUE ILEAL PROTEIN AND AMINO ACID DIGESTIBILITIESa(%) IN THE EXPERIMENTALDIETS
meal
CanOla meal
Protein
90.7b
73.5d
95.1b 91.1b 90.6b
Amino acids Indispensable Arginine Histidine Isoleucine Leucine
Lysine Methionine Phenylalanine Threonine Tryptophan Valine Dispensable Alanine Aspartic acid Cysteine Glutamic acid Glycine Proline Serine Tyrosine
go.@ 92.e 93 .Sb 87.1' 88.@ 88.3b 89.3bc 89Sb 90.Sb 86.2c
903 S5.Sb 88.3b 93.8b 95.3'
Barley
SE
88.8b
80.8'
1.6
84.4d 81.3' 75.od 77.4d 76.4d 84.8' 74.9d 71.1' 68.8' 74.ld
90.1'
84.Zd
1.7
S8.Sb
82.1'
90.9b 91.4b 85.4' 94.4b 92.6b
84.1' 85.2'
1.0 1.0
80.3' 73Ad 71.4d 85.2d 70.8*
88.1b
68.4b
81.1d 83 .4d
Wheat
90.2b
tE1.6~ 91.6b 89.1b 92.ob 97.ob 76.9bc 77.9b 94.6b 100.lb
82.6'
.9 1.8
87.V
1.2
83.4' 87Sb 69.4' 87.4'
1.8 1.9 3.0 1.0
79.6' 83.5' 87.5' 91.8' 63.2d 53.5'
1.5 1.4 1.3 .7 3.5 6.9 1.3 1.4
88.8'
95.5'
'Indirectly calculated, assuming 12.7 g endogenous protein per kilogram of DM intake and the amino acid composition of endogenous protein to be similar to that observed in pigs fed a protein-free diet and simultaneously administered with amino acids parentally (de Lange et al., 1989b). b3c,dValues in the s a r m row followed by different superscript letters differ (P< .OS).
having to enter the main blood circulation. This implies that the 'SNenrichmentexcess in amino acids, which argactually used for the synthesisof endogenous protein secretions, is lower than the excess measured in the TCA-soluble fraction of the blood. As a result, the contribution of endogenous to total protein in the gastro-intestinaltract is underestimated. Urea-N present in the TCAsoluble fraction of the blood also contributes to the "N-enrichment excess in the TCA soluble fraction. The "N-enrichment excess in urinary N usually was higher than that in the TCA-soluble fraction of blood (Figure 1). This implies that the "N-enrichment excess in urea was probably higher than in the free amino acids in the blood, which probably also resulted in an underestimation of the actual contribution of endogenous to total protein in the gastro-intestinal tract. The "N-enrichment excess in digesta, expressed as a percentage of the excess in the TCAsoluble fraction of blood, shows the relative contribution of endogenous to total protein in ileal digesta. These relative contributionsdid not
differ between d 7 and d 9 of the infusion period (P > .lo). Although no plateau was reached in the "N-enrichment excess in the different N pools, no differencewas observed in the relative contribution of endogenous protein to total protein in ileal digesta between d 7 and d 9 of the infusion periods. The real protein digestibilities therefore were calculated from these results. The amount of endogenous protein in ileal digesta varied between 25.5 and 30.5 g/kg DM intake (Table 6). These values were much higher than those observed in a previous experiment in which pigs were fed a protein-free diet while amino acids were simultaneously administered i.v. (de Lange et al., 1989) or the values observed in studies in which pigs were fed a protein-free diet (Wuensche et al., 1987; de Lange et al., 1989a). The quantities observed in the present study also were larger than those in studies in which alternative methods were used to determine the recovery of endogenous protein at the distal ileum in pigs: approximately 17.6 and 19.3 g per kg DM intake in pigs fed a barley-soybean
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Item
4 16
DE LANGE ET A L
TABLE 6. ENDOGENOUS PROTEIN IN ILEAL YJGESTAOF PIGS FED PROTEIN-CONTAINING DIETS AS DETERMINED WITH THE N-ISOTOPEDILUTION TECHNIQUE Item ~
Endogenous protein g per kg DM intake 96 of Total CP g per 100 g CPintake
Canda meal
m e a t
Barley
SE
25.5
30.5 53.9 18.0b
27.4 94.5. 19.1b
27.7 81.1b 24.7'
2.4 3.0 1.7
~~
84.6b
13.7b
8*b*c*dValua in the same row followed by different superscript letters differ (P < .OS).
meal diet (Zebrowska et al., 1982) and a fish meal-wheat diet (Simon et al., 1987), respectively. Zebrowska et al. (1982) calculated the contribution of endogenous to total protein in ileal digesta from the amino acid composition of dietary, endogenous and total protein in ileal digesta assuming that the amino acid composition of endogenous protein was constant and that the true or real digestibilities were similar for all amino acids. The last assumption, in particular, can be questioned because feedstuffs contain a variety of different proteins. The amino acid composition and the real ileal digestibilities of these proteins may vary. Simon et al. (1987) used an alternative 15~-isotope dilution technique in which proteincontaining diets were fed to pigs that were previously labeled with "N by feeding ["~~ammonium carbonate and lam ammonium chloride and in which the "N-enrichment excess in endogenousprotein was assumed to be similar to that in urinary N. Because the "N-enrichment excess in urinary N was higher than that in the TCA-soluble fraction of the blood in the resent study, it can be questioned whether the %-enrichment excess in urinary N is a valid indicator for that in endogenous protein. Only when the microbes present in the gastro-intestinal tract preferentially incorporate N originating from urea into microbial protein and when the conhibution of microbial N to total N in ileal digesta is larger than was previously estimated (Dierick et al., 1986), would the "Nenrichment excess in urine provide a better estimate of the enrichment excess in endogenous protein than that in the TCA-soluble fraction of the blood. In addition, differences in diet composition may contribute to the differences in the recovery of endogenous protein at the distal ileum in the present study and in studies by Zebrowska et al. (1982) and Simon et al. (1987). The present study showed that the indirectly
P
calculated true protein digestibilities underestimate the real protein digestibilities (Figure 2). The relatively low real protein digestibility in the CM diet (84.1%, Table 7) is probably related to its high fiber content (Bell, 1984). Components such as lignin, pectins, tannins and cellulose in CM may interfere with the digestion of protein in this feedstuff (Bell, 1984). Furthermore, the real protein digestibility in the barley diet was lower than 100% (94.296,Table 7). Eggum and Christensen (1975) showed that tannins, which are present in barley, may bind to protein (amino acids) and thereby affect the digestion and (re-) absorption of both endogenous and exogenous protein. Krawielitzki et al. (1977), in studies with rats, showed a linear relationship between the amount of dietary protein and the excretion of endogenous protein in feces. suggesting that the recovery of endogenous protein should be presented in units other than grams/kilogram DM intake, especially if the protein contents of the diets vary. Differences still were apparent (P c .05) between
EXlApparent
O T r u e (conv)
m R e a l ('5N)
Figure 2 Apparent, hue and real ileal protein digestibilities in the experimentaldiets.
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~~
Soybean meal
417
REAL ILEAL PROTEIN DIGESTIBILITIES IN PIGS TABLE 7. REAL ILEAL PROTEIN AND AMINO ACID DIGESTIBILITIEe (40) IN THE EXPERIMENTALDIETS
Item
Amino acids Indispensable Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Dispensable Alanine Aspartic acid Cysteine Glutamic acid Glycine proline Serine Tyrwine
Canola meal
Wheat
Barley
SE
97.9
84.ld
99.e
94.2'
1.0
98.Zb 94.6b 95.4c 949
90.2'
97.1b 94.7b
93.3c 90.7' 95.v 94.3c 97.7' 97.9
1.1 .9 1.2 1.1 1.2 1.2 1.8 3.3 3.0 1.7
96.6' 999 9 2 1' 101.w 93.1b %.gC
97.V 95.7' 97.9b 94.9 97.3b %.4b 100.2' 104.4'
86.7d 82.9d 84.9 M.od 91.8d 83.9d 87.2d 76.9' 84.ld 92.3* 85.3d 83.5'
91.v
85.P 77.9b 92.3d 99.7'
99.9 98Ab I01 .7b 102.2b 100.3b 118.2b W.4b 1O3.e l02.P l06.2b 103.Sb 100.3b 94.3b 83.7b 105.lb l16Sb
92.5' 113.3b 81.5' 100.lbc
96.9
1.4
1 02.6b 102.5b 97.4c 86.9c
1.8 21 .8 24 6.3 1.3 2.4
6o.v
104.9b 114.9b
'Directly determined real protein digestibilities using the "N-isotope dilution technique. The real amino acid digestibilities are estimated from the apparentprotein and amino acid digestibilities. the real protein digestibilities and the amino acid composition of endogenous protein as observed in pigs fed a protein-free diet and simultaneously administered with amino acids parentally (de Lange et al.. 1989b). b~c*d.eValues in the same row followed by different superscript letters differ (Pc .Os>.
diets if the recovery of endogenous protein was expressed as grams/l00 g CP intake (Table 6). The recovery was highest for the barley (24.7 g/lOO g CP intake) and lowest for the SBM diet (13.7g/lOogCPintake). Intheory,theseresults suggest that, if the four feedstuffs would have been compared at a similarprotein, barley would have resulted in the largest endogenous protein recoveries. More studies are necessary to identify the factors in these feedstuffs that are responsible for the observed differences in endogenous p r e tein recoveries. The aforementioned considerations also illustrate that difficulties may arise in the interpretation of apparent protein digestibilities, especially when the protein contents of the experimental diets vary and when different p r e tein sources are included in one diet (Imbeah et al., 1988). The N-isotope dilution technique only measures the recovery of total endogenous protein in ileal digesta or feces, not the recovery of the individual endogenous amino acids. In order to
calculate the real ileal amino digestibilities, the amino acid composition of endogenous protein was assumed to be similar to the amino acid composition of endogenous protein observed in a previous experiment in which a protein-fiee diet was fed to pigs while amino acids were administeredsimultaneously i.v. (de Lange et al., 1989b, Table 7). For some of the amino acids, especially in the wheat diet, the calculated real amino acid digestibilities exceeded 100%.Because the content of some of the amino acids in wheat was very low (Table 3) and the real protein digestibility was extremely high (Table 7), a small overestimation in the content of these amino acids in endogenous protein may result in overestimationof md amino acid digestibilities. Measuring the "N-enrichment excess in the individual amino acids as opposed to N in blood plasma and digesta may provide a better estimate of the real amino acid digestibilities. These results, however, indicate that the real amino acid digestibilitiesare higher than the true digestibil-
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Prorein
SOY-
meal
418
DE LANCE ET AL.
ities determined in conventional digestibility studies (Tables 5 and 7). Implications
Literature Cited
Alpers, D. H. 1972.Protein synthesis in intestinal mucosa: the effect of route of administration of precursor amino acids. J. of Clin. Invest 51:167. AOAC. 1980. Official methods of analyses (12th Ed.). Association of Official Analytical Chemists, Washington, DC. Bell, J. M. 1984. Nutrients and toxicants in rapeseed meal: A review. J. Anim Sci. 5 8 9 6 . Carlson, K. H. and H. S. Bayley. 1970. Nitrogenandamino acids in the feces of young pigs receiving a p t e i n free diet and diets containing graded levels of soybean meal or casein. 1. Nutr. 100:1353. de Lange, C.F.M., W. C. Sauer, R. Mosenthin and W. B. Souffrant. 1989a. The effwt of feeding different protein-free diets on the recovery and amino acid composition of endogenous protein collected from the distal ileum and feces in pigs. J. Anim. Sci. 67:746. de Lange, C.F.M.. W. C. Sauer and W. B. Souffrant. 1989b. The effect of protein status of the pig on the recovery and amino acid composition of endogenous protein in digesta collected from the distal ileum. J. Anim Sci. 67:755. Dierick, N. A., I. J. Vervaeke, J. A. Decuypere and H. K. Henderickx. 1986. Influence of the gut flora and of some growth-promoting feed additives on nitrogen metabolism in pigs. 11. Studies in vivo. Livest. prod. Sci. 14177. Eggum, B. 0. and K. D. Christenson. 1975. Influence of tannin on protein utilization of feedstuffs with special reference to barley. In: Breeding for Seed Im provement Using Nuclear Techniques. p 135. International Atomic Energy Agency, Vienna Fenton, T.W. and M. Fenton. 1979. An improved procedure for determination of chromic oxide in feed and feces. Can. J. Anim Sci. 59:631. Harvey, W. R. 1960. Least squan: analyses of data with unequal subclass numbers. USDA publ. ARS 2@8, Washington, DC. Imbeah, M.,W. C. Sauer and R. Mosenthin. 1988. The prediction of the digestible amino acid supply in
York. Tanksley, E D. and D. A. Knabe. 1984. Ileal digestibilities of amino acids in pig feeds and their use in formulating diets. In: W. Haresign and D.J.A. Cole (Ed.) Recent Advances in Animal Nutrition. p 75. Butterwarths, London. Waterlow. J. C., P. J. Garlick and D. J. Millward. 1978. Protein tllmover in mammalian tissues and in the whole body. North Holland, Amsterdam. Weirich, W.E., J. A. Will and C. W. Crumpton. 1970. A technique for placing chronic indwelling catheters in swine. J. Appl. Physiol. 28:117. Wuensche, J., U. Hermann, M. Meinl, U. Hennig, E Kreienbring and P. Zwien. 1987. Einfluss exogener Faktoren auf die praezaekaleNaehrstoff und Amine saeurenresorption ermittelt an Schweinen mit IleoRektal-Anastomosen. Arch. Tieremaehr. 37:745. Zebrowska, T., 0. Simon, R. Muenchmeyer, E. Wolf, €1. Bergner and H. Zebrowska 1982. Flow of endogenous and exogenous amino acids along the gut of pigs. Arch. Tierernaehr. 32431.
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The 15N-isotopedilution technique was used to determine the recovery of endogenous protein at the distal ileum in pigs. Endogenous protein was much higher than previously determined in studies in which protein-free diets were fed. The differences in apparent protein (and amino acid) digestibilities between the feedstuffs seemed U, be affected more by differences in the endogenous protein (and amino acid) recoveries than by differences in real protein (and amino acid) digestibilities. Studies on factors that affect the recovery of endogenous protein in ileal digesta and real amino acid digestibilitiesusing the "Nisotope dilution techniquedeserve more attention in future research.
barley-soybean meal ox canola meal diets and pan=tic secretions in pigs. J. Anim Sci. 66:1409. Kmwielitzki, K., T. Volker, S. Smulikowska, H. D. Bock and J. Wuensche. 1977. Weitex Untersuchungen zum Multikomptment-Modell des Protein-Stoffwechsels. Arch. Tieremaehr. 27:609. Kreienbring, E and I. Wuensche. 1976. Darlegung anwendbare Methoden zur Proteinhydrolyse und Aminosauerenbestimmung. Tag. Ber. Akad. Landwirtsch. Wiss. GDR (Berlin) 142:27. Matthews, D. E., E. B. Ben-Galimand D. M. Bier. 1979. Determination of stable isotopic enrichment in individual plasma amino acids by chemical ionizationmass spectrometry. Anal. Chem 51:80. NRC. 1979. Nutrient Requirements of Swine. (8th Rev. Ed.) National Academy Press, Washington, DC. NRC. 1988. Nuuient Requirements of Swine. (9th Rev. Ed.) National Academy Press, Washington, DC. Sauer, W. C., H. Jorgensen and R. Benins. 1983. A modified nylon bag technique for determining apparent digestibilities of protein in feedstuffs for pigs. Can. J. Anim Sci. 63233. Sauer, W.C. and L. Ozimek. 1986. Digestibility of amino acids in swine: results and theirpractical application. A =view. Livest. Prod. Sci. 15367. Simon, O., H. Bergner and I. G. Partridge. 1987. Estimation of endo enous N proportions in ileal digesta and faeces in kN-labelled pigs. Arch. Tierernaehr. 10851. Simon, O., T. Zebrowska, H. Bergner and R. Muenchmeyer. 1983. Investigations on the pancreatic and stomach secretions in pigs by means of continuous infusion of 14Camino acids. Arch. Tiexrnaehr. 33:9. Souffrant,W. B.. B. Darcy-Vrillon, T. Coning, J. P. Laplace, R. Koehler, G. Gebhardt and A. Rerat. 1986. Recycling of endogenous nitrogen in the pig. Arch. Tieremaehr. 36269. Souffrant, W. B., R. Koehler, R. Matkowitz, G. Gebhardt and H. Schmandke. 1981. Ernaehrungsphysie logische Untersuchungen an Schweinen zur beurteilung von modifierten Roteinen. Arch. Tieremaehr. 3 1675. Steel, R.G.D. and J. H. Tome. 1980. Principles and Prp cedures of Statistics. McGraw-Hill Book Co., New
