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* Department of Animal and Avian Sciences, University of Maryland, College Park, MD 20742, and Bovine Functional Genomics Laboratory, Animal and Natural Resources Institute, US Department of Agriculture-ARS, Beltsville, MD 20705
3 To whom correspondence should be addressed. Email: bbequett{at}umd.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • sheep • gastrointestinal tract • amino acid • urea • glucose
In ruminants, metabolism by the gastrointestinal tract (GIT)4 represents the single largest metabolic fate of amino acids (AAs), glucose, and other energy substrates in the body. Depending upon the AA, the GIT metabolizes 5–100% of the AA supply disappearing from the small intestines, and 50–100% of intestinal disappearance of glucose (1–3). The nutritional importance of GIT metabolism is well known, yet there are still important aspects of GIT metabolism that remain largely unresolved. With regard to AA metabolism, it could be hypothesized that, at a minimum, net metabolism of essential AAs by the GIT occurs at a fixed rate and an amount relative to their compositions in the endogenous proteins lost from the GIT. Thus, once this "service cost" is satisfied, absorption of AAs across the GIT should approximate to 100% of their additional intestinal supplies. Current information suggests that this may not be the case, however, particularly with regard to certain nonessential AAs (4–9).
Net absorption of AAs by the small intestines of ruminants has been determined from the product of net arteriovenous difference and blood flow measurements across the mesenteric-drained viscera (MDV). Most often, however, net metabolism by the whole GIT is determined from measurements across the portal-drained viscera (PDV), which represents the sum of MDV plus rumen and hind-gut metabolism. This measure accounts for intestinal metabolism as well as that occurring by the rumen and hind-gut tissues, the latter of which derive AAs almost exclusively from the arterial blood supply (1). Although this arterial use of AAs by the PDV is considered a consequence of GIT metabolism, this arterial use of AAs represents potential competition with peripheral tissues (e.g., muscle, mammary gland). Based on PDV net flux measurements (dairy cows: (3,4,10); sheep: (1)) net recovery of infused casein, or free AAs, or absorbed essential AAs, is generally found to be lowest for the branched-chain AAs (BCAA: leucine, valine and isoleucine; 10–62%), followed variously by methionine (33–69%), histidine (39–92%), phenylalanine (36–96%), lysine (30–66%), tryptophan (31–88%) and threonine (41–63%). By contrast, intestinal supplies of the nonessential AAs glutamine and glutamate have been found to be almost completely metabolized by the GIT, even when the supplies of these AAs are increased (4–9). These studies examined PDV nutrient use in response to only one level of protein, AAs or feed intake, and so it has not been possible to establish response relations of intestinal supply and net absorption of individual AAs. In consequence, it is not known whether GIT metabolism occurs at a fixed or variable rate for individual AAs, and nor is it known whether this GIT metabolism ultimately leads to differential supplies of AAs to the liver, and beyond to peripheral tissues, for anabolic use.
The aim of this study is to answer 2 central questions regarding AA utilization by the ruminant GIT: 1) are certain AAs preferentially utilized, and 2) are certain AAs metabolized at fixed or variable rates relative to changes in their intestinal supply? A secondary aim relates to observations that the GIT of ruminants net metabolizes the intestinal supply of glucose (5,11). For this reason, the net balance of glucose across the GIT is often negative. In this respect, a goal was to determine whether glucose utilization is reduced or unaffected by changes in the intestinal supply and GIT utilization of AAs.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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5 cm from the ileo-cecal vein junction at the most proximal point where the venous drainage from proximal small intestine enters the mesenteric arcade (see 12). The tip of the distal mesenteric vein catheter was positioned 30 cm upstream of the proximal catheter for infusion of the blood flow marker para-aminohippuric acid (PAH). A catheter (polyvinyl chloride, 2.4 mm i.d. x 4.0 mm o.d.) was also inserted into the proximal duodenum, the tip 10 cm from the pylorus, for infusion of casein. From 2 wk postsurgery, feed (Table 1) was delivered in equal portions every 2 h via an automatic feeder. Sheep were fed to 2 times energy maintenance intake (10.9 MJ/d, 80 g dry matter/kg BW0.75) a pelleted ration that was adequate in energy (calculated 10.2 MJ metabolizable energy/kg dry matter) but which was low in protein (10.4% crude protein, dry matter) and provided only 60% of the metabolizable protein intake (75 g/d) required to support 300 g of gain per day (13).
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Tracer infusion and blood sampling. On day 4 of casein infusions, a temporary catheter (polyvinyl chloride, 0.8 mm i.d., 1.20 mm o.d.) was inserted (10 cm) into a jugular vein. On the last day, a sterile solution containing PAH (0.1 mol/L) and heparin (235 kIU/L) was infused (8 h, 20 mL/h) into the distal mesenteric vein catheter, and into the jugular vein a sterile solution containing [1-13C]leucine (1.5 g/L) and [6-2H2]glucose (5 g/L) was infused (8 h, 20 mL/h). At the same time, [methyl-2H3]leucine (30 mg/h) was infused into the duodenum along with water or casein infusions. Over the final 4 h, blood samples were continuously withdrawn (5 mL/h) over 1-h periods from the artery, proximal mesenteric vein, and portal vein by peristaltic pump and collected into sealed syringes submerged in an ice bath. Each syringe was mixed by gentle hand rolling, and the plasma separated by centrifugation (1000 x g for 15 min at 4°C).
Plasma amino acid and urea concentration. These were determined by isotope dilution with gas chromatography-mass spectrometry (GC-MS) as previously described (14). To a known weight (0.5 g) of fresh plasma was added an equal known weight of a solution containing 0.2 mg hydrolyzed [U-13C]algae protein powder (99 atoms %; Martek Biosciences), 100 nmol [indole-2H5]tryptophan, 200 nmol [5-15N]glutamine, 25 nmol [methyl-2H3]methionine, and 3 µmol [15N2]urea, and the samples stored frozen (–20°C). Thawed samples were deproteinized by addition (1 mL) of sulfosalicylic acid (15% w:v), the supernatant desalted by cation (AG-50, H+ form) exchange, and AAs and urea eluted with 2 mol/L NH4OH followed by water. For urea analysis, 20 µL of this eluate was dried under a stream of N2, and urea converted to the t-butyldimethylsilyl derivative prior to GC-MS (HP 5973N Mass Selective Detector, Agilent). The remaining eluate was lyophilized to dryness, and AAs converted to their t-butyldimethylsilyl derivative. Under electron impact mode, the following ions (m/z) were monitored: urea 231, 233; alanine 260, 263; glycine 246, 248; valine 288, 293; isoleucine 302, 308; proline 286, 291; methionine 292, 295; serine 390, 393; threonine 404, 408; phenylalanine 234, 242; aspartate 302, 304; glutamate 432, 437; lysine 300, 306; histidine 440, 446; glutamine 168, 169; tyrosine 302, 304; and tryptophan 244, 249. For leucine isotope enrichment and concentration, ions at 302, 303 ([1-13C]leucine), 305 ([2H3]leucine), and 308 ([13C6]leucine, internal standard) were monitored. Calibration curves were generated from gravimetric mixtures of labeled and unlabeled AA. For leucine, correction was also made for the spillover of [1-13C]leucine (m/z 303) into [2H3]leucine (m/z 305), and spillover of [2H 3]leucine into [13C6]leucine (m/z 308). All enrichments were expressed as atoms percent excess (APE) relative to background natural abundance.
Glucose concentration and enrichment. To a known weight (0.150 g) of fresh plasma was added an equal known weight of a solution containing 0.6 mmol [13C6]glucose. The aldonitrile pentaacetate derivative of glucose was formed (15), and ions at 242, 244 ([2H2]glucose) and 247 ([13C6]glucose) monitored by GC-MS under electrical impact mode. This derivative of glucose loses carbon-1 under electron impact to yield m/z 247, which corresponds to the internal standard [13C6]glucose. Calibration curves were generated, and corrections made for spillover of m/z 244 ([2H2]glucose) to m/z 247 ([13C6]glucose). Enrichments are expressed as APE above background.
PAH concentration. Plasma concentrations of PAH were determined in duplicate using 0.25 g plasma and employing gravimetric procedures (16).
Calculation of net fluxes of amino acids. Net absorption or removal of an AA by the mesenteric (MDV; mostly small intestines) and portal (PDV; whole GIT) drained viscera was calculated as the product of plasma flow (F; [kg/(kg BW · h)]) and plasma venoarterial concentration difference (µmol/kg plasma) as appropriate. The incremental recovery by the MDV and PDV of individual AAs infused as casein into the duodenum was determined from the slope of the regression of net flux rate against duodenal casein–AA infusion rate [µmol/(kg BW · h]. The intestinal digestibility of casein was assumed to be 100% (17).
Calculation of leucine fluxes.
Rate leucine of appearance (Leu Ra) was calculated from arterial plasma leucine enrichment (EA) when the leucine tracer was infused into the jugular vein ([13C]leucine; systemic Leu Ra) or into the duodenum ([2H3]leucine; whole body Leu Ra):
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where EI is the APE of the leucine tracer infused ([2H3] or [1-13C]), and IR is isotope infusion rate per kg BW per h. The difference between whole body Leu Ra and systemic Leu Ra is leucine removed by the gut and liver during its first pass. Fractional first-pass arterial utilization of leucine by the MDV or PDV was calculated from jugular infusion of [13C]leucine:
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where [A] is arterial and [V] is MDV or PDV concentration of leucine as appropriate. First-pass arterial utilization by the MDV or PDV was calculated as:
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where F is plasma flow rate for the MDV or PDV as appropriate. Fractional first-pass intestinal utilization of leucine was computed from [2H3]leucine tracer balance across the PDV, corrected for [2H3]leucine, recycled, and sequestered on second-pass by the PDV from the arterial circulation (i.e., [13C]leucine arterial removal):
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where IR is the rate of [2H3]leucine infusion into the duodenum.
Calculation of glucose utilization.
Net removal of plasma glucose by the MDV and PDV was calculated as above for AAs. Glucose rate of appearance (gluconeogenesis, glucose recycling, and absorption) was calculated as described for Leu Ra. First-pass arterial utilization of glucose by the MDV and PDV was calculated as:
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where E is the enrichment (APE) of [2H2]glucose in the artery (A) and vein (V; MDV or PDV).
Statistical analysis.
For all data, ANOVA assumptions were checked prior to analysis. Data were analyzed by 3-way ANOVA for a 4x4 Latin square design using the MIXED procedure of SAS (version 8.0, SAS Institute), in which the infusion level was the fixed effect and sheep and experimental period are random effects. The following linear mixed model was used:
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where Yijk is the observed value for the kth sheep, the jth period and the ith treatment, 
is the grand mean, Ti is the treatment effect for the ith treatment, Rj is the period effect for the jth period, Ck is the sheep effect for the kth sheep, and 
ijk is the random error associated with Yijk. When a significant treatment effect was detected, means were compared using Tukey-Kramer multiple comparison test. Backward stepwise regression was performed in which a third-order model was tried first, and, if not significant, the analysis was repeated with a lower-order model until significance was reached. The 95% CI were calculated for the slopes of the regression equation. Significance of differences between first-pass utilization and net absorption was assessed by Student's t test. Data are presented as least square means ± SEM and differences were considered significant at P 
0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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75 g/d. The MDV measurements for one sheep were omitted due to catheter tip misalignment noted postmortem, and for another sheep the PDV data were omitted for the last 2 treatment periods (35 and 105 g casein/d) due to PDV catheter blockage. Plasma concentrations. Casein infusion significantly increased (P < 0.05) arterial concentrations (Table 2) of the BCAA, methionine, phenylalanine, and proline, whereas glycine concentration decreased (P < 0.05). Plasma urea also increased (P < 0.001) with level of casein infusion, with values doubling between the control and the 105 g/d casein infusion level.
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net absorption ÷ casein–AA infusion) was computed based on the slope of regressing net AA absorption against the casein–AA infusion rate for that AA (see Fig. 1). The 95% CI were generated to test for unity, i.e., a slope of 1 = 100% recovery of the infused AA. Net MDV absorption (Table 4) of all essential and nonessential AAs, except for glutamate and glutamine, was linearly (P < 0.05) related to their casein–AA infusion rates. The incremental efficiency of absorption across the MDV fell within a narrow range (0.87–1.05) for all essential AAs, excluding tryptophan. For the nonessential AAs, values ranged from 0.46 for aspartate to 1.79 for alanine. Because of the wide 95% CI associated with each AA measurement, however, none of the slopes were significantly different from unity for the MDV (small intestines). Thus, recovery of AA across the MDV was not different from 100%.
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For the MDV and PDV, net removal of urea from plasma was not affected by casein infusion although, numerically, the values were higher. The correlation of urea removal with plasma urea concentration was significant for the MDV (P = 0.0379, r = 0.90) and PDV (P = 0.0124, r = 0.93).
Leucine metabolism. Rate of casein infusion increased (P < 0.01) systemic and whole body leucine Ra (Table 7). As a proportion of whole body leucine Ra, the MDV accounted for 0.16 (± 0.059), whereas casein infusion increased the proportion (0.33–0.60 ± 0.060, P < 0.033) of whole body leucine Ra partition to the PDV. Although net absorption of leucine by the PDV increased with casein infusion, there was also increased (P < 0.05) first-pass arterial utilization of leucine by the PDV. For the MDV, net absorption of leucine increased with casein infusion, but there was no change in the rate of first-pass arterial utilization. Fractional first-pass intestinal utilization was computed from PDV, rather than MDV, flux measurements to avoid introduction of errors due to site of luminal tracer infusion and location of the downstream MDV sampling catheter. Fractional first-pass intestinal utilization of leucine was higher (Student's t test, P < 0.05) than fractional first-pass arterial utilization, but neither was altered by casein infusion.
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0.38 of whole body glucose Ra, whereas the MDV accounted for a smaller but increasing (0.11–0.24) proportion in response to casein infusion. Because of the higher rates of first-pass arterial utilization of plasma glucose, the net balances of glucose across the PDV and MDV were negative (i.e., arterial utilization > net absorption) and these were not significantly affected by casein infusion. With first-pass arterial utilization of glucose by the PDV (Student's t test, P < 0.001) and MDV (Student's t test, P < 0.0011), both exceeded net glucose removal, indicating that glucose was absorbed from the small intestines only to be removed from the blood on second pass.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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It was also important that our measurements of GIT metabolism were made over a wide range of intestinal protein supplies, spanning from marginal to above the estimated metabolizable protein requirements for maintenance and growth of sheep with this body weight. This was achieved by infusing casein into the duodenum at 4 levels (0, 35, 70, and 105 g/d). The highest level of protein supply was predicted to raise total protein supply (diet + casein infusion) to 110% of the metabolizable protein supply required to support gain of 300 g/d for sheep of this size (13). Furthermore, to acquire a more representative estimate of the efficiency of AA absorption, net fluxes for each AA were plotted against their respective casein–AA infusion rates (Fig. 1 and Tables 5 and 6). Regression slopes were analogous to the net efficiency of absorption above the basal level of protein intake in this study. CI (95%) were then computed from the slope relationships, and tests against unity (i.e., 100%) were performed for each AA.
We highlight 3 observations from the slopes of the regression analysis. First, the net absorption data for each essential AA best fit a linear relation (P < 0.005; Fig. 1 and Tables 5 and 6) in response to casein infusion. We had hypothesized that, because the level of metabolizable protein intake (75 g/d) of the basal ration was low relative to growth requirements, net AA requirements of the GIT may not have been met and, in consequence, net absorption would not increase until perhaps the 70 g casein infusion level. Even with the limited observations in this study (see Fig. 1), there appears to be no indication of an inflection in net absorption, but this will need to be further confirmed at lower protein intakes and with a larger number of observations.
Second, the 95% CI of the slopes indicated that essential AA absorption by the MDV (small intestines) was effectively 100% (i.e., slope = 1) whereas absorption of the BCAA by the PDV was significantly <100% (i.e., slope <1). Also approaching significance were the slopes for threonine (slope 0.75, 95% CI 0.36–1.10) and lysine (slope 0.78, 95% CI 0.55–1.05) for the PDV. We interpret these data as a demonstration that the small intestines (MDV) were in net balance with respect to essential AA utilization, irrespective of the rate of intestinal protein supplies above the maintenance requirement. The net efficiency of absorption of the BCAA by the PDV was <100% (slope ranges: 0.60–0.66, 95% CI 0.31–0.91) across the protein infusion levels with, in consequence, greater net metabolism (e.g., lost as protein into the GIT or catabolized) of the BCAA as intestinal protein supply increased. In fact, for leucine, as representative of the BCAA, the majority of the increased use by the GIT occurred from the arterial blood supply (Table 7), mainly by the non-MDV tissues (i.e., rumen and hind gut). MacRae et al. (1,2) made similar observations in sheep (35 kg BW) that were fed alfalfa pellets (15.6% crude protein) at 2 levels of intake (maintenance vs. 1.5 x maintenance). In that report, recoveries of the essential AAs in the MDV was 100% (mean 106%, range 74–131%), whereas recoveries by the PDV were significantly less (mean 69%, range 45–96%) than the disappearance of the AAs from the small intestines (duodenum to ileum).
Third, for both glutamate and glutamine, net absorption by the MDV and PDV were far less than their intestinal supplies and net absorption was not increased by casein infusion. We find it of interest that the BCAA, glutamate, and glutamine were utilized by the whole GIT in greater amounts as the supply of protein to the small intestines increased. The primary fates of AAs utilized by the GIT are catabolism and incorporation into constitutive and secretory proteins (6,17). Except for the threonine-rich mucoproteins (20), the AA composition of GIT protein is not likely to fluctuate. In this respect, we consider it unlikely that the higher removals of the BCAAs, glutamate, and glutamine by the GIT relates to their increased incorporation into GIT proteins (and endogenous losses). If the additional removal of these AAs had been directed toward GIT protein synthesis, we should have also observed a corresponding increase in the net removal of other essential AAs, which we did not observe. The more likely explanation is that these AAs were catabolized either by luminal microbes or by gut tissues either from the luminal, arterial, or both aspects of the GIT.
The BCAA have been shown to be oxidized by the GIT of ruminants (19) and considerable evidence indicates extensive catabolism of glutamate and glutamine by the GIT of ruminants and monogastric species (4–9). For both leucine and glutamate, oxidation by the GIT fluctuates in direct relation to intestinal protein supply (dairy cattle: (21); piglets: (8)). To date, it remains unresolved whether AA catabolism occurs directly by the GIT tissues or by microbes in the intestinal lumen. The present data indicate nearly complete metabolism of glutamate and glutamine by the MDV tissue (small intestines), but it was not possible to determine the extent of luminal vs. arterial use. However, based on leucine kinetic data, most of the increased use of leucine by the GIT occurred from the arterial supply in response to casein infusion. Therefore, we suggest that the GIT tissues, but not luminal microbes, were responsible for metabolism of the BCAA.
Numerous studies in rats (see 9) and piglets ((6) and (22)), under conditions of adequate food intake, have shown that AAs are the major substrates (50–70%) contributing to oxidative energy generation by the intestinal mucosa. By contrast, glucose contributes relatively less (30–40%) (23). Similar information is not complete for ruminant animals and, to date, investigations in vivo have been limited to glutamate and glutamine (24), leucine (25,26) and lysine, methionine, and phenylalanine (19). Given that the GIT of the sheep appears to metabolize greater amounts of the BCAA, glutamate, and glutamine as protein supply is increased, it was logical to consider whether metabolism of other nutrients would be reciprocally reduced, namely, glucose. The majority of glucose supplied to the ruminant GIT is from the arterial blood, and so it is commonly observed that the net balance of glucose across the MDV and PDV is negative. There is, of course, some glucose available to the small intestinal lumen from dietary starch escaping the rumen and from bacterial polysaccharides (27). It is apparent from the comparison of unidirectional and net removals of glucose (Table 8) that glucose was presented to the small intestinal lumen and absorbed into the MDV and PDV blood drainages (i.e., unidirectional > net removal).
Net removal and first-pass arterial utilization of glucose by the GIT remained constant across the levels of casein infusion, suggesting that glucose use by the PDV was not spared by the additional protein supply. This is perhaps not surprising for 2 reasons. First, Van der Schoor et al. (8) observed that glucose oxidation by the PDV of piglets remained the same at 2 levels of protein intake (9.6 vs. 21.6 g/(kg · d)). And, second, catabolism of BCAA carbon skeletons occurs mainly via acetyl-CoA, and for these AAs to spare glucose from complete catabolism necessitates that glucose is also metabolized via this route. In a study conducted with isolated sheep rumen epithelial and small intestinal mucosal cells (28), we observed that glucose contributed to only 1–5% of acetyl-CoA flux, despite the fact that glucose made a significant contribution to pyruvate flux (8–38%). Therefore, it would not be expected that increased metabolism of the BCAA by the GIT would lead to sparing glucose from complete catabolism. In contrast, we have shown that sheep rumen and intestinal cells partially catabolize glucose to pyruvate, and thence to alanine (28), and that net production of alanine by intestinal cells is increased in the presence of glucose (29). In this connection, the high and increased net release of alanine by the PDV with casein infusion may reflect greater partition of glucose toward alanine synthesis and release.
In summary, AA and glucose metabolism by the MDV and PDV was examined in growing sheep in response to incremental increases in protein supply to the small intestines that spanned the range of metabolizable protein supplies from marginal to well above requirements for maintenance and growth. The efficiency of absorption above maintenance of all the essential AAs, except the BCAA, was effectively 100%, and this remained fixed at levels of protein supply above maintenance. By contrast, the efficiency of absorption the BCAA, glutamate, and glutamine were <100%, and for glutamate and glutamine high rates of net metabolized by the GIT were maintained even at upper levels of intestinal protein supply. Despite the excess metabolism of these AAs, glucose utilization by the GIT was not altered, and therefore not spared by AA supply. The basis for the greater metabolism of the BCAA, glutamate, and glutamine with increased intestinal protein supply, remains uncertain. In practical terms, the nearly complete catabolism of these nonessential AAs, and to some extent aspartate, by the GIT necessitates that they be synthesized de novo from carbon skeletons and nitrogen that likely derive from absorbed essential AAs.
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FOOTNOTES |
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2 Presented in part at the annual meetings of the Federation of Animal Science Societies, July 25–29, 2004, Saint-Louis, MO [El-Kadi SW, Sunny NE, Oba M, Owens SL, Bequette BJ. Fractional removal of amino acids by the small intestines and whole gastrointestinal tract of sheep remains constant across levels of protein supply] and July 24–28, 2005, Cincinnati, OH [El-Kadi SW, Baldwin RL VI, Sunny NE, Owens SL, Bequette BJ. Postruminal protein infusion increases leucine use by the gastrointestinal tract of sheep while glucose utilization remains unchanged].
4 Abbreviations used: AA, amino acid; APE, atom percent excess; BW, body weight; GC-MS, gas chromatography-mass spectrometry; GIT, gastrointestinal tract; MDV, mesenteric-drained viscera; PAH, p-amino-hippuric acid; PDV, portal-drained viscera.
Manuscript received 7 October 2005. Initial review completed 13 November 2005. Revision accepted 8 February 2006.
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