J. Anim Sci. 2006. 84:1749-1760. doi:10.2527/jas.2005-558
© 2006 American Society of Animal Science
Biochemical and morphological developments are partially impaired in intestinal mucosa from growing pigs fed reduced-protein diets supplemented with crystalline amino acids1
F. Guay*,2,
S. M. Donovan
and
N. L. Trottier*,3
* Department of Animal Science, Michigan State University, East Lansing 48824
and
Food Science and Human Nutrition, University of Illinois, Urbana 61801
 |
Abstract
|
|---|
The objective of this study was to determine if a reduction in dietary CP, with partial replacement of the intact protein with crystalline AA (CAA), would alter growth, morphology, and free or peptide-bound AA concentrations of intestinal mucosa in growing pigs. Twenty-four barrows (37.0 ± 1.5 kg of BW) were fed 1 of 4 diets for 24 d: 16.1% CP with no CAA, or 12.8, 10.1, or 7.8% CP (analyzed values, as-fed) containing CAA. As CP decreased, CAA were gradually increased to meet requirements on a true ileal digestible basis. Pigs were euthanized 2 h postmeal on d 24, and mucosal samples from duodenum, jejunum, and ileum were collected. Reducing dietary CP decreased ADG, G:F, and final weight (linear, P < 0.05). With reduced dietary CP, mucosal protein concentration decreased in the jejunum (quadratic, P < 0.05) and tended to decrease in the ileum (linear, P = 0.062). Reduction of the dietary CP concentration from 16.1 to 7.8% tended to decrease the crypt depth (linear, P < 0.10) and decreased villus width (linear, P < 0.05) in duodenum and jejunum mucosa but did not reduce villus height or villus surface area in any regions of the small intestine. In the duodenum, a reduction in dietary CP increased free Lys, Met, and Thr (linear, P < 0.05) and peptide-bound Lys and Thr (quadratic, P < 0.10). In the jejunum, reducing CP decreased free Cys (linear P < 0.05) and tended to decrease free Asn and His (linear, P < 0.10) and peptide-bound His (quadratic, P = 0.061) and Ile, Leu, and Val (linear, P < 0.10). In the ileum, reducing CP decreased free Asn, Ser, Tyr, Arg, His, Phe (linear, P < 0.05), and Leu (linear, P = 0.054) and peptide-bound Gly and Ser (linear, P < 0.05) and tended to decrease peptide-bound Ile, Leu, Phe, Val (linear, P < 0.10), and Lys (linear P < 0.05). In conclusion, reduced-CP diets supplemented with CAA lead to a reduction in growth performance, associated with biochemical and morphological modifications of the intestinal mucosa.
Key Words: amino acid • intestinal mucosa • pig • protein
|
INTRODUCTION
|
|---|
In growing pig diets, crystalline AA (CAA) can be used as replacement for protein-bound AA to improve protein quality and reduce fecal N excretion (Kerr and Easter, 1995
; Le Bellego et al., 2002; Otto et al., 2003). However, feeding diets severely reduced in CP led to reduced growth rate and N retention in several studies (Gomez et al., 2002; Zervas and Zijlstra, 2002; Otto et al., 2003), despite dietary inclusion of CAA to restore the indispensable AA. In pigs, feeding low CP diets without AA supplementation decreases total protein mass of portal-drained viscera and reduces rate of mucosal protein synthesis in the jejunum (Sève et al., 1993; Ebner et al., 1994; Wykes et al., 1996). Minimal level of enteral nutrients is necessary to sustain normal mucosal growth and to maximize intestinal protein accretion (Burrin et al., 2000; Stoll et al., 2000). It is unknown whether reduced-CP diets supplemented with CAA can maintain growth and integrity of the intestinal mucosa. The intestinal tract may be limited in its ability to utilize CAA and consequently may depend on a minimum dietary level of AA in peptide-bound form to sustain intestinal mucosal integrity. This question is based on the notion that absorption of AA by mucosal cells occurs partially in the form of di- and tripeptides (Leibach and Ganapathy, 1996) in diets containing intact ingredients as a source of protein-bound AA.
The study presented here addresses in part whether or not dietary inclusion of CAA affects intestinal mucosal functions. We hypothesized that CAA used as replacements for limiting indispensable protein-bound AA in reduced-CP diets cannot sustain normal intestinal growth and morphology when dietary CP is severely restricted. The objectives of this study were to determine 1) if partial replacement of protein-bound AA with CAA can maintain normal gut growth and morphology; and 2) whether or not the impaired intestinal growth and morphology are associated with reduction in peptide-bound AA in the intestinal mucosa.
|
MATERIALS AND METHODS
|
|---|
Experimental Diets
Four diets were formulated to contain corn and soybean meal as the source of protein-bound AA (Table 1). Analyzed CP concentration values were 16.1, 12.8, 10.1, and 7.8% for 15% CP, 12% CP + CAA, 9% CP + CAA, and 6% CP + CAA diet, respectively. The analyzed CP concentrations are used throughout the manuscript to identify the respective diets. The 16.1% CP diet was formulated using corn and soybean meal to meet the true ileal digestible requirement for Lys for a 50-kg barrow with a predicted lean growth rate of 325 g/d (NRC, 1998). Ratios between corn and soybean meal were increased to achieve 12.8 and 10.1% CP. In the 12.8% CP diet, Lys, Thr, and Trp were limiting; thus, crystalline L-Lys·HCl, L-Thr, and L-Trp were included to meet the true ileal digestible requirement for those AA (NRC, 1998). In the 10.1% CP diet, additional AA were limiting, including Met, Ile, Val, and Phe; thus, crystalline sources of those AA were included. The 7.8% CP diet contained corn as the sole source of protein-bound AA. In the 7.8% CP diet, all indispensable AA were limiting and thus were included in their crystalline form. Nutrient composition, including analyzed and calculated AA composition of each diet, is presented in Table 2.
View this table:
[in this window]
[in a new window]
|
Table 2. Analyzed total and calculated true ileal digestible AA composition and nutritional values of diets (as-fed basis)
|
|
Animals and Housing
Animals were managed throughout the study in accordance with requirements of the Michigan State University All University Committee on Animal Use and Care. Twenty-four barrows (Yorkshire-Landrace; 37.0 ± 1.5 kg of initial BW) were selected from 6 litters and transferred to individual metabolism crates (1.5 x 1.5 m). Growing pigs were housed in a temperature-controlled room kept at 20 ± 2°C with constant lighting. Growing pigs received 2.0 kg of the 16.1% diet daily during the first 4 d and were then assigned according to their initial BW to the 4 dietary treatments in a randomized complete block design with 6 pigs per treatment. Feed was provided daily in 2 equal meals (0800 and 1600) for 24 d. Feeding level was adjusted weekly to provide 5 times the ME required for maintenance (106 kcal/kg of BW0.75), and pigs had free access to water at all times. Pigs were weighed at the beginning of the study (d 0), on d 7, 14, and 21, and before euthanasia on d 24. Feed intake was measured daily.
Tissue Collection
On d 24, pigs were fed 2 h before euthanasia. Pigs were first sedated with an i.m. injection (0.029 mL/kg of BW) of 250 mg of zolazapam, 250 mg of tiletamine diluted in 2.5 mL of ketamine solution (100 mg/mL), and 2.5 mL of xylazine-100 (100 mg/mL). After sedation, pigs were euthanized with an intravenous administration of pentobarbital (86.27 mg/kg of BW).
The entire small intestine was quickly removed and freed from the mesentery. The segment from the pyloric anthrum to 100 cm caudal to the pyloric anthrum was considered the duodenal region; the segment extending from the cecum to 100 cm cranial to the cecum was considered the ileal region; and the segment between the duodenum and ileum was considered the jejunal region (Yen, 2001). For each segment, 5 cm from the middle section was removed for histological analysis, and the adjacent 20 cm was used for biochemical analyses.
The 20-cm intestinal segments were rinsed thoroughly with ice-cold saline solution (0.9% NaCl), opened lengthwise, blotted dry, and weighed. The mucosa was scraped from the underlying tissue using a glass slide, immediately transferred into liquid N, and then stored at –80°C until analysis. The intestinal segments were then weighed without mucosa to estimate mucosal weight for each 20-cm intestinal segment. Mucosal weight for the 20-cm segment was used to express the biochemical composition and enzymatic activities on the basis of intestinal length (i.e., mass or activity, respectively, per unit of intestinal length).
The 5-cm intestinal segments for light microscopic analysis were immediately perfused with ice-cold saline solution to remove debris. Each of these segments was opened, pinned villus-side up, and fixed in 10% formalin solution (Sigma-Aldrich Co., St. Louis, MO) for 24 h. Each segment was then cut into 4 pieces and maintained in 10% formalin solution until examined. The entire procedure was completed within 35 min after euthanasia.
Biochemical Analyses
The mucosa from each segment was homogenized with a tissue homogenizer (Ultra-Turrax T27, IKA-La-bortechnik, Stenfer, Germany) using a 0.4:2.5 (g/mL) ratio of tissue and phosphate-buffered saline EDTA (0.05 M NaPO4, 2.0 M NaCl, 2 x 10–3 M EDTA, pH 7.4) for DNA analysis and the same ratio of tissue and distilled water for RNA, protein, and free and peptide-bound AA. Total proteins were measured by the method of Lowry et al. (1951) using a detergent-compatible protein assay (BioRad Laboratories, Hercules, CA) and BSA as standards. Deoxyribonucleic acid content was estimated by a fluorometric assay (Labarca and Paigen, 1980), and RNA content was evaluated by colorimetric assay (Volkin and Cahn, 1954).
Mucosal AA concentrations were determined by HPLC, using precolumn derivatization with PITC (Pierce Inc., Rockford, IL), coupled to a Waters solvent delivery system (Waters Corporation, Milford, MA). A Pico-Tag column (3.9 x 300 mm, Waters Corporation) was used. A specific gradient elution was performed at 46°C, using Pico-Tag eluent 1 and 2 (Waters Corporation) as the mobile phase, with a flow rate of 1.0 mL/min. Norleucine (40 µL, 1.25 mM) was added to 200 µL of homogenate and 1 mL of trifluoroacetic acid/methanol (1:10 ratio). The homogenate was centrifuged (5,000 x g for 10 min). For free AA, the supernatant (155 µL) was evaporated to dryness with a centrifuge evaporator (Heto-Holten AS, Gydevang, Denmark) for 3 h. Redrying solution (20 µL of sodium acetate:methanol:triethylamine, 2:2:1) was added to each sample. Samples were dried with a centrifuge evaporator (Heto-Holten AS) for 2 h. Derivatizing reagent (20 µL of methanol:water:triethylamine:PITC, 7:1:1:1) was added to each sample. Methanol (20 µL) was also added to each tube. After a reaction time of 10 min, samples were dried by evaporative centrifugation (Heto-Holten AS) for 3 h. To remove possible residues of PITC, methanol (20 µL) was again added to each sample. Samples were then dried for 3 h by evaporative centrifugation (Heto-Holten AS) and rehydrated with diluent solution (200 µL of PIC-Tag solution:methanol, 4:1). Then, samples (20 µL) were directly injected into the column using a Water 2690 separation module (Waters Corporation). Amino acids were detected after HPLC separation with a Waters 486 absorbance detector (Waters Corporation) at 254 nm. Peak area analyses for AA were performed by Millennium Chromatography Manager Software (Waters Corporation).
Concentrations of peptide-bound AA were determined according to the method proposed by Rémond et al. (2000) for sheep plasma. Specifically, 500 µL of supernatant, from the mucosal homogenate used to determine free AA concentration, was added to 500 µL of water and filtered through a 3,000 molecular weight cut-off filter (Centricon-YM3, Millipore, Bedford, MA) at 7,500 x g for 4 h at 4°C. The filtrate was evaporated to dryness by evaporative centrifugation (Heto-Holten AS) for 6 h. The residue was resuspended in 1 mL of 6 N HCl and hydrolyzed at 115°C for 24 h. The hydrolysate was filtered through a 0.45-µm syringe filter and evaporated to dryness for 6 h. The residue was resuspended in 0.5 mL of methanol and directly analyzed as described for free AA. Peak analysis of Asp and Glu in hydrolyzed samples could not be done with exactness; thus accurate determination of these AA concentrations was not possible. Peptide-bound AA were derived from the difference between posthydrolysis and prehydrolysis (free AA) AA concentrations.
Enzyme Analyses
Peptidase activities were measured spectrophotometrically at 410 nm as described by Sangild et al. (1995). Briefly, frozen intestinal tissue was minced finely, extracted in 1% Triton X-100 (6 mL/g of tissue), homogenized with a tissue homogenizer (Ultra-Turrax T27) on ice, and centrifuged at 13,000 x g for 1 h. The supernatant was used for enzyme analysis. Aminopeptidase N activity in mucosa (dilution 1:500) was measured using 10 mM L-alanine-4-nitroanilide (Sigma-Aldrich Co.) as the substrate and 50 mM Tris-HCl, pH 7.3, as the buffer at 37°C for 20 min. Dipeptidyl peptidase IV activity in mucosa (dilution 1:100) was measured using 15 mM L-glycyl-L-proline-4-nitroanilide (Sigma-Aldrich Co.) as the substrate and 50 mM Tris-HCl, pH 8.0, as the buffer at 37°C for 20 min. Results were compared using a specific blank obtained with a substrate and specific buffer mix.
Morphometric Analysis
For morphometric analysis, fixed intestinal samples were embedded in paraffin, sectioned at 6 µm, and stained with eosin and hematoxylin. Histochemical sections were observed with a Nikon Optiphot-2 microscope (Nikon, Melville, NY), and digital images were captured using Image-Pro Plus software, version 3.0 (Media Cybernetics, Silver Spring, MD). Villus height, midvillus width, and crypt depth were measured in a minimum of 10 well-defined villi and crypts in the duodenum, jejunum, and ileum (Houle et al., 1997).
Feed Analysis
Feed samples were finely ground using a sample mill (Cyclotec 1093, Foss Tecator, Sweden). Total N was determined with a N analyzer (Fp-2000, Laboratory Equipments Corporation, St. Joseph, MI) using EDTA as a calibration standard. Samples of feed were hydrolyzed in 6 N HCl at 110°C for 24 h, before analysis by reverse-phase HPLC as previously described for analysis of free AA. Norleucine was added as an internal standard before hydrolysis. Determination of Trp and sulfur AA concentrations was performed at the Missouri Agricultural Experiment Station Chemical Laboratories (University of Missouri—Columbia). Briefly, Trp was determined by colorimetric assay after enzymatic digestion. Sulfur AA were determined according to AOAC (2000). Starch analysis was performed using an adaptation of the method of Karkalas (1985). Briefly, samples were gelatinized with 50% NaOH, hydrolyzed with HCl and amylase, and absorbance was read at 450 nm.
Statistical Analysis
Data were analyzed as a randomized complete block design using the MIXED procedure of SAS (SAS Inst. Inc., Croy, NC). The model was: Yijk = µ + Bi + Lj + Dk + eijk, where Yij = the dependent variable, µ = mean of the variable, Bi = initial BW (block), Lj = litter, Dk = dietary treatments, and eijk = residual error. Relationships between dietary CP concentrations and animal growth, intestinal growth, or intestinal AA concentrations (free and peptide form) were determined using orthogonal polynomials (linear, quadratic, and cubic). Significant differences and tendencies for differences were determined at P values of < 0.05 and < 0.1, respectively.
|
RESULTS
|
|---|
Body Weight Gain
Dietary CP concentration did not affect ADFI during the experimental period (Table 3). However, reducing dietary CP resulted in a linear decrease in overall ADG, G:F, and final weight (P < 0.05).
View this table:
[in this window]
[in a new window]
|
Table 3. Growth performance of growing pigs fed a corn-soybean meal-based diet and reduced-CP diets supplemented with crystalline AA (CAA)1
|
|
Intestinal Mucosal Composition and Morphology
Reducing dietary CP concentration did not affect intestinal and mucosal weights across the different regions of the small intestine (Table 4) except for ileal weight, which tended to be heavier in pigs fed the 7.8% diet compared with that in pigs fed other diets (linear, P = 0.087). In the duodenum, there were no differences among dietary treatments in intestinal protein, RNA, and DNA contents (Table 4). In the jejunum, mucosal protein concentration decreased (quadratic, P < 0.05), and mucosal DNA concentration tended to decrease (linear, P = 0.085; Table 4) as dietary CP decreased. In the ileum, only protein concentration (linear, P = 0.062) tended to decrease as dietary CP decreased. Reduction of dietary CP concentration from 16.1 to 7.8% tended to decrease (linear, P < 0.10) the crypt depth (Table 4) and decreased (linear, P < 0.05) villus width in the duodenum and jejunum. In the jejunum, villus height/crypt depth (V/C) ratios increased with reduction of dietary CP concentration (linear, P < 0.05). However, villus height and villus surface area in duodenum and jejunum were not affected by reduction of dietary CP concentration. In the ileum, intestinal morphology responses did not differ among dietary treatments, but villus height of pigs fed 12.8, 10.1, and 7.8% diets was numerically reduced by 29, 9, and 18% CP, respectively, compared with that of pigs fed the 16.1% CP diet (linear, P = 0.125). Reducing dietary CP decreased dipeptidyl peptidase IV and aminopeptidase N activities in the duodenum (linear and cubic for Figure 1 and 2, respectively, P < 0.05) but not in the jejunum or ileum.
View this table:
[in this window]
[in a new window]
|
Table 4. Intestinal composition of growing pigs fed a corn-soybean meal-based diet and reduced-CP diets supplemented with crystalline AA (CAA)1
|
|
View larger version (16K):
[in this window]
[in a new window]
|
Figure 1. Dipeptidyl peptidase IV activity in the duodenum, jejunum, and ileum of growing pigs. Growing pigs were fed corn-soybean meal diets containing varied protein concentrations and crystalline AA (CAA): 16.1% was a corn-soybean meal diet without CAA, and the 12.8, 10.1, and 7.8% were reduced-CP diets supplemented with CAA to meet true ileal digestible requirements (NRC, 1998). *Linear effect of dietary treatment, P < 0.05. Values are means ± SEM for 6 pigs.
|
|
View larger version (19K):
[in this window]
[in a new window]
|
Figure 2. Aminopeptidase N activity in the duodenum, jejunum, and ileum of growing pigs. Growing pigs were fed corn-soybean meal diets containing varied protein concentrations and crystalline AA (CAA): 16.1% was corn-soybean meal diet without CAA, and the 12.8, 10.1, and 7.8% were reduced-CP diets supplemented with CAA to meet true ileal digestible requirements (NRC, 1998). *Cubic effect of dietary treatment, P < 0.05. Data are means ± SEM for 6 pigs.
|
|
The reduction of dietary CP concentration led to lower liver weight (Table 4
) compared with that of pigs fed the 16.1% diet (linear, P = 0.072). Relative liver weight (liver weight/BW) of pigs fed the 10.1 and 12.8% diets was reduced compared with that of pigs fed the 16.1% diet but was not affected in pigs fed the 7.8% diet (quadratic, P = 0.093).
Intestinal Mucosal Concentrations of Free and Peptide-Bound Amino Acids
Duodenum.
Reduction of dietary CP concentration increased the concentration of free Ala (linear, P = 0.062) and Gly (quadratic, P < 0.05; Table 5). Concentrations of other free dispensable AA did not differ among dietary treatments. Reduction of dietary CP concentration increased concentrations of free Lys, Met, and Thr (linear, P < 0.05) but had no effect on other free indispensable AA. Concentrations of total free AA in duodenal mucosa of pigs fed the 16.1% diet were 25% lower compared with that of pigs fed reduced-CP diets (quadratic, P = 0.095). In the duodenum, concentration of peptide-bound Lys and Thr increased with reduction of dietary CP, reaching maximal concentrations in the 12.8 and 10.1% diets (quadratic, P < 0.05 for Lys, and P = 0.091 for Thr).
View this table:
[in this window]
[in a new window]
|
Table 5. Duodenal mucosal concentrations of free AA and peptide AA of growing pigs fed a corn-soybean meal-based diet and reduced-CP diets supplemented with crystalline AA (CAA)1
|
|
Jejunum.
Reduction in dietary CP concentration had no effect on the majority of free dispensable and indispensable AA concentrations but decreased Cys (linear, P < 0.05) and tended to reduce concentrations of free Asn and His (linear, P < 0.10), and Gly (quadratic, P = 0.091; Table 6). Reduction of dietary CP concentration tended to lower concentrations of peptide-bound His, Ile, Leu, Val, and total indispensable AA (linear, P < 0.10; except for His, quadratic, P = 0.061).
View this table:
[in this window]
[in a new window]
|
Table 6. Jejunal mucosal concentrations of free AA and peptide AA of growing pigs fed corn-soybean meal-based diet and reduced-CP diets supplemented with crystalline AA (CAA)1
|
|
Ileum.
Concentrations of free Asn, Ser, and Tyr decreased with reduction in dietary CP concentration (linear, P < 0.05; Table 7). Reduction of dietary CP concentration from 16.1 to 7.8% resulted also in a linear decrease in concentrations of free Arg, His, and Phe (linear, P < 0.05) and a tendency to reduce Leu, Trp (linear, P < 0.10), and Val concentrations (quadratic, P = 0.062). Concentration of free Thr increased with reduction in dietary CP concentration (quadratic, P < 0.05). Total and indispensable AA concentration did not decrease with reduced-CP diets. For peptide-bound AA, concentrations of Gly and Ser decreased with reduction of dietary CP concentration (linear, P < 0.05). Concentrations of 5 indispensable peptide-bound AA (His, Ile, Leu, Lys, Phe, Val) decreased with reduction of dietary CP concentration (linear, P < 0.10; except for His, quadratic, P = 0.062 and for Lys, linear, P < 0.01). The concentration of peptide-bound Tyr tended to increase with reduction of dietary CP (quadratic, P = 0.098).
View this table:
[in this window]
[in a new window]
|
Table 7. Ileal mucosal concentrations of free AA and peptide AA of growing pigs fed a corn-soybean meal-based diet and reduced-CP diets supplemented with crystalline AA (CAA)1
|
|
|
DISCUSSION
|
|---|
In growing pigs fed diets reduced in CP, normal growth and N retention can be maintained when CAA are included. However, whole-body growth cannot be sustained when dietary CP is severely restricted (Gomez et al., 2002; Zervas and Zijlstra, 2002; Otto et al., 2003). In this study, moderate reduction in CP from 16.1 to 12.8% with inclusion of CAA reduced overall growth performance by 10%, whereas severe reduction to 10.1 and 7.8% reduced overall ADG and G:F by approximately 16 and 30%, respectively, despite inclusion of CAA that met true ileal digestible AA requirements. Thus, we asked if the limitation of whole body growth could in part be explained by changes in intestinal mucosal growth, morphology, and AA profile (free and peptide-bound), which in turn may have an impact on the absorption and use of nutrients by peripheral tissues.
In the duodenum, despite reduction of crypt depth and villus width, mucosal DNA and protein contents remained unchanged as CP concentration decreased, agreeing with Sève et al. (1993), who reported no change in duodenal protein synthesis rate and protein concentration in pigs fed reduced-CP diets. In parallel, greater contents of free Lys, Met, and Thr, with no change in peptide-bound AA contents with decreasing dietary CP indicate that AA availability, free and peptide-bound, to the brush-border membrane of the duodenal mucosa was not limiting. Unlike duodenal mucosa, protein metabolism of the jejunal and ileal mucosa was modified in response to feeding reduced-CP + CAA diets as indicated by a decrease in protein concentration. Similarly, nutritional protein deficiency without supplementation of indispensable AA reduces protein synthesis rate and protein content (Wykes et al., 1996; Dudley et al., 1997; 2001). Feeding stimulates protein synthesis in the jejunal mucosa in the neonatal pigs (Burrin et al., 1995; Davis et al., 1996), indicating that nutrients (e.g., AA) absorbed through the intestinal mucosa can control directly or indirectly protein synthesis in visceral tissues. On the other hand, parenteral infusion of AA increases protein synthesis in the liver and pancreas of piglets (Davis et al., 2002) but results in lower protein synthesis in the jejunal mucosa (Dudley et al., 1998; Bertolo et al., 1999), which in turn is associated with significant reduction in mucosal-free AA concentrations (Bertolo et al., 2000). In this study, jejunal mucosa of pigs fed reduced-CP + CAA diets had lower concentrations of peptide-bound indispensable AA, but free AA contents, except for Cit and Orn, remained largely unaffected. Daenzer et al. (2001) showed that dietary protein-bound Lys and Leu are used more efficiently for distal small intestinal mucosal protein synthesis than dietary free Leu and Lys. The fact that free Lys and Thr are absorbed more rapidly than protein-bound dietary Lys and Thr as reported by Yen et al. (2004) may reflect a difference in mucosal use and, consequently, portal appearance. Thus in this study, the decrease in protein-bound AA availability to the lower small intestinal mucosa contributed to the decrease in jejunal mucosa protein content.
In contrast to the duodenum and jejunum, decreases in both peptide-bound and free AA associated with decreasing dietary CP were observed in the ileal mucosa. A parallel tendency for reduction in villus height was observed, but like in jejunum, no significant reduction in villus surface area, aminopeptidase N, and dipeptidyl peptidase IV activities were noted. Thus, whereas total AA availability to the ileal mucosa may have to a small extent limited the maintenance of villus morphological integrity, absorption surface area and enzymatic activity of the ileal and also duodenal and jejunal mucosa remained unaffected. The metabolic events governing how AA directly or indirectly affect intestinal morphology are unknown. Dietary AA are utilized for protein synthesis, but also as an important energy source for the intestinal mucosa, and are precursors for intestinal synthesis of glutathione, purine, and pyrimidine nucleotides, and AA (Reeds et al., 1996; Bertolo et al., 1999; Stoll et al., 1999). In fact, only 18, 21, 18, and 12% of the total first-pass metabolism of Lys, Leu, Phe, and Thr, respectively, were recovered in the mucosal intestinal protein of piglets (Stoll et al., 1998). For dispensable AA, the proportional appearance of dietary AA in portal blood indicates that the portal-drained viscera utilize almost completely enteral Glu, Asp, and Gln (Reeds et al., 1996; Stoll et al., 1998, 1999). In our study, the mucosal concentration of major energy substrates (free Glu, Gln, Asp) were not affected in spite of reduction of dietary ileal dispensable AA concentrations from 80.5 to 26.5% between 16.1 and 7.8% diets, indicating that other substrates may compensate to some extent in supplying energy to the intestinal mucosa. For instance, Van der Schoor et al. (2001) have shown that visceral Leu and Glu oxidations were largely suppressed and glucose oxidation increased by 50% of the total visceral CO2 production during reduced-protein intake in pigs.
Growth performance was limited in particular when protein was severely reduced. However, some of the intestinal morphological and biochemical measurements indicated that changes were already occurring with only moderate reduction (16.1 to 12.8%) in dietary CP. Additionally, the relationship between dietary CP reduction and some of the intestinal composition measurements was curvilinear, with increasing values with extreme protein reduction (16.1 to 7.8%). Thus, the physiological significance of alteration in intestinal morphology and biochemical processes remains unclear. Admittedly, some measurements were more variable than others; hence the number of animals may have limited acquisition of a complete picture of the relationship between dietary protein-bound AA with CAA and intestinal physiological processes. Nonetheless, the data are novel and indicate that some physiological changes occurred with replacement of limiting indispensable protein-bound AA with a free crystalline source within a specific dietary CP range (16.1 to 10.1%), despite dietary provision of CAA that met ileal digestible requirements for growth. Intestinal use of free AA may thus be limited, as previously discussed.
In conclusion, partial replacement of protein-bound AA with CAA in reduced-CP diets led to lower mucosal protein content in the jejunum and ileum, but not in the duodenum, indicating a modification of protein metabolism in the lower proximal and distal small intestine. Correspondingly, peptide-bound AA concentration in both the jejunal and ileal mucosa decreased, and the large majority of free AA concentrations decreased in the ileal mucosa only. However, villus surface area, and aminopeptidase N and dipeptidyl peptidase IV activities in jejunal and ileal mucosa were not reduced, indicating that the absorption surface for nutrients remained unaltered. Furthermore, ileal mucosal morphology was largely unaffected despite a decrease in total AA (free and peptide-bound) availability. In contrast, whereas few biochemical changes occurred, some morphological changes were observed in the duodenal mucosa, despite that peptide-bound AA concentrations remained unaltered and the free AA concentrations increased. Results indicate that changes in mucosal morphology in the proximal small intestine, and protein content in the lower proximal and distal intestine occurred concurrently with a decrease in growth and feed efficiency. However, the results also indicate the AA form, i.e., free or peptide-bound, has little impact on mucosal morphology, and as such it should be recognized that other nutritional or nonnutritional factors might have contributed to the mucosal morphological changes observed in this study. Conversely, results indicate that mucosal protein content may depend on peptide-bound AA rather than free AA and may thus contribute to a decrease in global protein accretion under reduced-CP diets. Finally, the change in AA profile in intestinal mucosa as seen in this study, and the portal appearance profile of AA as demonstrated by Yen et al. (2004) in pigs fed reduced-CP diets with CAA, may have important consequences on postgut AA use by peripheral tissues, and consequently on the global protein accretion.
|
IMPLICATIONS
|
|---|
Reducing dietary protein-bound amino acid concentrations led to alteration of protein metabolism in the mid to lower small intestine, despite inclusion of indispensable crystalline amino acids. Nevertheless, the absorption surface for nutrients across all intestinal segments might be unaffected. The profiles of peptide-bound and free amino acids were altered in the jejunum and ileum in reduced crude protein diets. These modifications may impair amino acid use by peripheral tissues, and thus reduce global protein synthesis. Finally, mucosal decrease of amino acids in animals fed reduced crude protein diets supplemented with crystalline amino acids merits further investigations with regard to the long-term impact on the maintenance of intestinal morphology.
|
Footnotes
|
|---|
1 This study was supported by the National Pork Board, the Michigan State Agricultural Experiment Station, and a postdoctoral fellowship from the Fond pour la Formation des Chercheurs et l’Aide à la Recherche (FCAR) du Québec. The authors wish to thank J. Moore, K. Ochonicky, J. Pérez Laspiur, P. Ku, and C. Wickens for their assistance in animal handling, tissue collection, and laboratory analysis and Duane E. Ullrey for reviewing the manuscript. 
2 Current address: Département des sciences animales, Faculté des sciences de l’agriculture et de l’alimentation, Pavillon Paul-Comtois 4201, Université Laval, Québec, QC G1K 7P4, Canada.
3 Corresponding author: trottier{at}msu.edu
Received for publication September 28, 2005.
Accepted for publication January 24, 2006.
|
LITERATURE CITED
|
|---|
AOAC. 2000. Official Methods of Analysis. 16th ed. Assoc. Off. Anal. Chem., Arlington, VA.
Bertolo, R. F. P., C. Z. L. Chen, P. B. Pencharz, and R. O. Ball. 1999. Intestinal atrophy has a greater impact on nitrogen metabolism than liver by-pass in piglets fed identical diets via gastric, central venous or portal venous routes. J. Nutr. 129:1045–1052.[Abstract/Free Full Text]
Bertolo, R. F. P., P. B. Pencharz, and R. O. Ball. 2000. Organ and plasma amino acid concentrations are profoundly different in piglets fed identical diets via gastric, central venous or portal venous routes. J. Nutr. 130:1261–1266.[Abstract/Free Full Text]
Burrin, D. G., T. A. Davis, S. Ebner, P. A. Schoknecht, M. L. Fiorotto, P. J. Reeds, and S. McAvoy. 1995. Nutrient-independent and nutrient-dependent factors stimulate protein synthesis in colostrum-fed newborn pigs. Pediatr. Res. 37:593–599.[Medline]
Burrin, D. G., B. Stoll, R. Jiang, X. Chang, B. Hartman, J. J. Holst, G. H. Greeley, and P. J. Reeds. 2000. Minimal enteral nutrient requirements for intestinal growth in neonatal piglets: How much is enough? Am. J. Clin. Nutr. 71:1603–1610.
Daenzer, M., J. Petzke, B. J. Bequette, and C. C. Metges. 2001. Whole-body nitrogen and splanchnic amino acid metabolism differ in rats fed mixed diets containing casein or its corresponding amino acid mixture. J. Nutr. 131:1965–1972.[Abstract/Free Full Text]
Davis, T. A., D. G. Burrin, M. L. Fiorotto, and H. V. Nguyen. 1996. Protein synthesis in skeletal muscle and jejunum is more responsive to feeding in 7- than in 26-day-old pigs. Am. J. Physiol. 270:E802–E809.[Medline]
Davis, T. A., M. L. Fiorotto, D. G. Burrin, P. J. Reeds, H. V. Nguyen, P. R. Beckett, R. C. Vann, and P. M. J. O’Connor. 2002. Stimulation of protein synthesis by both insulin and amino acids is unique to skeletal muscle in neonatal pigs. Am. J. Physiol. Endocrinol. Metab. 282:E880–E890.[Abstract/Free Full Text]
Dudley, M. A., P. A. Schoknecht, A. W. Dudley, L. Jiang, R. P. Ferraris, J. N. Rosenberger, J. F. Henry, and P. J. Reeds. 2001. Lactase synthesis is pretranslationally regulated in protein-deficient pigs fed a protein-sufficient diet. J. Nutr. 280:G621–G628.
Dudley, M. A., L. J. Wykes, A. W. Dudley, D. G. Burrin, B. L. Nichols, J. Rosenberger, F. Jahoor, W. C. Heird, and P. J. Reeds. 1998. Parenteral nutrition selectively decreases protein synthesis in the small intestine. Am. J. Physiol. Gastrointest. Liver Physiol. 274:G131–G137.[Abstract/Free Full Text]
Dudley, M. A., L. Wykes, A. W. Dudley, M. Fiorotto, D. G. Burrin, J. N. Rosenberger, F. Jahoor, and P. J. Reeds. 1997. Lactase phlorizin hydrolase synthesis is decreased in protein-malnourished pigs. J. Nutr. 127:687–693.[Abstract/Free Full Text]
Ebner, S., P. Schoknecht, P. J. Reeds, and D. G. Burrin. 1994. Growth and metabolism of gastrointestinal and skeletal muscle tissues in protein-malnourished neonatal pigs. Am. J. Physiol. 266:R1736–R1743.[Medline]
Gomez, R. S., A. J. Lewis, P. S. Miller, and H. Y. Chen. 2002. Growth performance, diet apparent digestibility, and plasma metabolite concentrations of growing pigs fed corn-soybean meal diets or low-protein, amino acid-supplemented diets at different feeding levels. J. Anim. Sci. 80:644–653.[Abstract/Free Full Text]
Houle, V. M., E. A. Schroeder, J. Odle, and S. M. Donovan. 1997. Small intestinal disaccharidase activity and ileal villus height are increased in piglets consuming formula containing recombinant human insulin-like growth factor-I. Pediatr. Res. 42:78–86.[Medline]
Karkalas, J. 1985. An improved enzymic method for the determination of native and modified starch. J. Sci. Food Agric. 36:1019–1027.[CrossRef]
Kerr, B. J., and R. A. Easter. 1995. Effect of feeding reduced protein, amino acid-supplemented diets on nitrogen and energy balance in grower pigs. J. Anim. Sci. 73:3000–3008.[Abstract]
Labarca, C., and K. Paigen. 1980. A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem. 1:344–352.
Le Bellego, L., J. Van Milgen, and J. Noblet. 2002. Effect of high temperature and low-protein diets on the performance of growing-finishing pigs. J. Anim. Sci. 80:691–701.[Abstract/Free Full Text]
Leibach, F. H., and V. Ganapathy. 1996. Peptide transporters in the intestine and the kidney. Annu. Rev. Nutr. 16:99–119.[CrossRef][Medline]
Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with folin phenol reagent. J. Biol. Chem. 193:265–275.[Free Full Text]
NRC. 1998. Pages 111–114 in Nutrient Requirements of Swine. 10th rev. ed. Natl. Acad. Press, Washington, DC.
Otto, E. R., M. Yokoyama, P. K. Ku, N. K. Ames, and N. L. Trottier. 2003. Nitrogen balance and ileal amino acid digestibility in growing pigs fed diets reduced in protein concentration. J. Anim. Sci. 81:1743–1753.[Abstract/Free Full Text]
Reeds, P. J., D. G. Burrin, F. Jahoor, L. Wykes, J. Henry, and E. M. Frazer. 1996. Enteral glutamate is almost completely metabolized in first pass by the gastrointestinal tract of infant pigs. Am. J. Physiol. 270:E413–E418.[Medline]
Rémond, D., L. Bernard, and C. Poncet. 2000. Free and peptide amino acid net flux across the rumen and the mesenteric and portal-drained viscera of sheep. J. Anim. Sci. 78:1960–1972.[Abstract/Free Full Text]
Sangild, P. T., H. Sjostrom, O. Noren, A. L. Fowden, and M. Silver. 1995. The prenatal development and glucocorticoid control of brush-border hydrolases in the pig small intestine. Pediatr. Res. 37:207–212.[Medline]
Sève, B., O. Ballèvre, P. Ganier, J. Noblet, J. Prognaud, and C. Obled. 1993. Recombinant porcine somatotropin and dietary protein enhance protein synthesis in growing pigs. J. Nutr. 123:529–540.[Abstract/Free Full Text]
Stein, H. H., S. W. Kim, T. T. Nielsen, and R. A. Easter. 2001. Standardized ileal protein and amino acid digestibility by growing pigs and sows. J. Anim. Sci. 79:2113–2122.[Abstract/Free Full Text]
Stoll, B., D. G. Burrin, J. Henry, H. Yu, F. Jahoor, and P. J. Reeds. 1999. Substrate oxidation by the portal drained viscera of fed piglets. Am. J. Physiol. 277:E168–E175.[Medline]
Stoll, B., X. Chang, M. Z. Fan, P. J. Reeds, and D. G. Burrin. 2000. Enteral nutrient intake level determines intestinal protein synthesis and accretion rates in neonatal pigs. Am. J. Physiol. Gastrointest. Liver Physiol. 279:G288–G294.[Abstract/Free Full Text]
Stoll, B., J. Henry, P. J. Reeds, H. Yu, F. Jahoor, and D. G. Burrin. 1998. Catabolism dominates the first-pass intestinal metabolism of dietary essential amino acids in milk protein-fed piglets. J. Nutr. 128:606–614.[Abstract/Free Full Text]
Van der Schoor, S. R., J. B. Van Goudoever, B. Stoll, J. F. Henry, J. R. Rosenberger, D. G. Burrin, and P. J. Reeds. 2001. The pattern of intestinal substrate oxidation is altered by protein restriction in pigs. Gastroenterology 121:1167–1175.[CrossRef][Medline]
Volkin, E., and W. E. Cahn. 1954. Estimation of nucleic acids. Methods Biochem. Anal. 1:287–303.[Medline]
Wykes, L. J., M. Fiorotto, D. G. Burrin, M. D. Rosario, M. E. Frazer, W. G. Pond, and F. Jahoor. 1996. Chronic low protein intake reduces tissue protein synthesis in a pig model of protein malnutrition. J. Nutr. 126:1481–1488.[Abstract/Free Full Text]
Yen, J. T. 2001. Digestive system. Pages 390–453 in Biology of Domestic Pig. W. G. Pond, and H. J. Mersmann, ed. Cornell Univ. Press, Ithaca, NY.
Yen, J. T., B. J. Kerr, R. A. Easter, and A. M. Parkhurst. 2004. Difference in rates of net portal absorption between crystalline and protein-bound lysine and threonine in growing pigs fed once daily. J. Anim. Sci. 82:1079–1090.[Abstract/Free Full Text]
Zervas, S., and R. T. Zijlstra. 2002. Effects of dietary protein and fermentable fiber on nitrogen excretion patterns and plasma urea in grower pigs. J. Anim. Sci. 80:3247–3256.[Abstract/Free Full Text]
This article has been cited by other articles:
|
|
|
|
 
A. Deglaire, P. J. Moughan, S. M. Rutherfurd, C. Bos, and D. Tome
Feeding Dietary Peptides to Growing Rats Enhances Gut Endogenous Protein Flows Compared with Feeding Protein-Free or Free Amino Acid-Based Diets
J. Nutr.,
November 1, 2007;
137(11):
2431 - 2436.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. N. Nortey, J. F. Patience, P. H. Simmins, N. L. Trottier, and R. T. Zijlstra
Effects of individual or combined xylanase and phytase supplementation on energy, amino acid, and phosphorus digestibility and growth performance of grower pigs fed wheat-based diets containing wheat millrun
J Anim Sci,
June 1, 2007;
85(6):
1432 - 1443.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Guay and N. L. Trottier
Muscle growth and plasma concentrations of amino acids, insulin-like growth factor-I, and insulin in growing pigs fed reduced-protein diets
J Anim Sci,
November 1, 2006;
84(11):
3010 - 3019.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
INTRODUCTION
