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Article

Replacement of Soybean Oil with Tallow in Rye-Based Diets without Xylanase Increases Protein Synthesis in Small Intestine of Broilers¹

Sven Dänicke^*2, Wolfgang Böttcher^*, Heinz Jeroch^*, Jens Thielebein and Ortwin Simon^**

^* Institute of Animal Nutrition and Planned Crop Storage, Agricultural Faculty, Martin-Luther-University Halle-Wittenberg, Germany; Institute of Animal Breeding, Animal Husbandry with Veterinary Surgery, Agricultural Faculty, Martin-Luther-University Halle-Wittenberg, Germany; and ^** Institute of Animal Nutrition, Free University of Berlin, Berlin, Germany

²To whom correspondence should be addressed.

	ABSTRACT

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We examined the effects of dietary fat type (10% of either soybean oil, S, or beef tallow, T)³ and xylanase supplementation (-, without; +, with 1 g of Avizyme 1300 per kg diet) in rye- based diets (56%) on tissue protein synthesis in male broilers. Birds were injected with a large flooding dose of a phenylalanine solution (150 mmol/L, 38 atom percentage excess [¹⁵ N] phenylalanine) and tissues were obtained after a 10-min incorporation period. [¹⁵ N]-enrichment in tissue free phenylalanine and tissue protein bound phenylalanine were measured by gas-chromatography mass-spectrometry and by gas-chromatography combustion isotope ratio mass-spectrometry, respectively in order to calculate tissue specific fractional rates of protein synthesis (k_s). The k_s (%/d) in (S-), (S+), (T-) and (T+)-fed birds were 56, 64, 84 and 61 (SEM = 3.7) in duodenum, 51, 52, 75 and 58 in jejunum (SEM = 3.1), 66, 67, 105 and 68 (SEM =7.0) in jejunal mucosa cells, 53, 56, 68 and 50 (SEM = 3.7) in ileum and 52, 45, 118 and 39 (SEM = 20.2) in pancreas, respectively. Significant fat, enzyme or interaction effects in these tissues were mainly caused by the elevated k_s in (T-)-fed birds which was closely associated with intestinal viscosity. We conclude that the effect of soluble nonstarch polysaccharides (NSP) and of NSP-hydrolyzing enzymes may be explained partially by modification in tissue protein synthesis of the intestinal tract.

KEY WORDS: • broilers • protein synthesis rate • rye • fat type • xylanase

	INTRODUCTION

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Variation in concentrations of soluble nonstarch-polysaccharides (NSP³ ) of wheat, triticale, barley and rye is correlated with in vitro cereal extract viscosity and to intestinal viscosity in broilers (Bedford and Classen 1992 , Bhatty et al. 1991 , Dusel et al. 1997 , 1998, Salih et al. 1991 ). Increases in small intestinal viscosity in broilers have been shown to reduce performance, nutrient digestibility and metabolizability of energy (e.g., Almirall et al. 1995 , Bedford and Classen 1992 , Salih et al. 1991 ) and to increase the small intestinal populations of some microbial groups (Dänicke et al. 1999a ,Vahjen et al. 1998 ). Furthermore, soluble NSP were shown to influence gut morphology and mucosal cell turnover. Feeding high concentrations of soluble NSP was found to increase the length, the absolute and relative weight of small intestine as well as to increase the rate of intestinal cell division in rats (Johnson and Gee 1986 , Johnson et al. 1984 ). Similar results have been observed in broilers by Langhout et al. (1999) , Simon (1998) and Viveros et al. (1994) .

Dietary fat concentration and quality may also contribute to changes in intestinal morphology as shown by Sagher et al. (1991) , who demonstrated that feeding of corn oil and olive oil significantly increased villus height compared to a low-fat control diet, whereas feeding of butter fat significantly decreased villus height.

The use of NSP-hydrolyzing enzymes as feed additives have been proven as effective tools in countering the gel-forming properties of soluble NSP (see Jeroch et al. 1995 , for review). However, the extent of response to enzyme was dependent on dietary fat type, the response in general being greater if the fat was of animal rather than plant origin (Dänicke et al. 1997a , 1997b ; Langhout et al. 1997 ; Smulikowska and Mieczkowska 1996 ).

Morphological and physiological changes in the intestine mediated by viscosity or by fat type might consequently be associated with intestinal protein synthesis. Intestinal protein synthesis itself plays such a large role in determining total protein metabolism, which includes endogenous nitrogen losses, of the animal that changes in its rate will influence overall nitrogen balance of the animal significantly. Hence, the aim of the study was to examine the effects of two fat types, S and T, differing mainly in their concentration of unsaturated fatty acids in a rye-based diet without or with addition of a NSP-hydrolyzing enzyme preparation on protein synthesis and protein growth in small intestinal tissues and other tissues.

	MATERIALS AND METHODS

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Diets and Animals.

Semipurified rye-based diets (56% dietary rye inclusion) were prepared either with 10% S or with 10% T and were fed without (-) or with (+) supplementation of a xylanase containing enzyme preparation (1 g/kg diet of Avizyme 1300; Finnfeeds International, Marlborough, United Kingdom). The resulting four experimental diets were calculated to be isonitrogeneous (Table 1 ) and were provided as pellets. In addition, diets were formulated to be isocalorically in order to minimize metabolizable energy-related regulation in feed and nutrient intake by the birds. Differences in metabolizable energy concentrations between T and S were compensated by dietary inclusion of starch or crystalline cellulose. Crystalline cellulose is not utilized by broilers (Scott et al. 1982 ); its metabolizable energy concentration is almost zero. In addition, it was shown that increasing cellulose concentration up to 120 g/kg diet does not influence endogenous N-secretions in cocks (Green 1988 ). Therefore, cellulose was used as a more or less inert feed compound.

Animal procedures.

Procedures were approved by the local Animal Care Committee (Regierungspräsidium Dessau, Sachsen-Anhalt, Aktenzeichen: 53a-42502/2–167).

Experimental procedures were carried out at 23, 26 and 29 d of age. At each time point, live weights were recorded after a 4-h period of feed withdrawal.

Measurement of fractional rates of protein synthesis (k_s) was performed at d 26 of age. Seven birds per treatment representing the average live weight of their groups were selected. Birds were injected with a large dose of a phenylalanine solution (150 mmol/L, 38 atom percentage excess [¹⁵ N] phenylalanine), prepared from 99.2 atom percentage [¹⁵ N] phenylalanine (Isotec, Miamisburg, OH), via wing vein at a rate of 1 mL/100 g live weight according to the procedure described by Garlick et al. (1980) . Birds were killed by cervical dislocation followed by decapitation exactly 10 min after injection. Mixed trunk blood was collected into heparinized test tubes and kept on ice prior to preparation of plasma. The small intestine with adhering pancreas and liver were quickly excised and spread out on a glass sheet placed on ice. The liver (without gall bladder) and pancreas were dissected, weighed and frozen in liquid nitrogen. The small intestine was divided into duodenum (pylorus to entrance of main pancreatic and biliary ducts), jejunum (from end of duodenum to Meckel’s diverticulum) and ileum (from end of jejunum to ileo-caecal junction). Each of these segments was further divided into three parts of a same length. The middle third was processed first. Digesta was removed by flushing each segment with ice-cold saline (9 g of NaCl/L). Segments were then opened longitudinally, excess saline was carefully blotted and weight was recorded. The middle third of each segment was frozen in liquid nitrogen and stored at -20°C until further analysis. The procedure for the jejunal third was modified in so far as this segment was additionally divided into two parts after final weighing. The proximal part was frozen and from the distal part mucosal cells were scraped off using a microscope slide prior to freezing. The whole procedure was finished within 2 min, and the exact time from killing to freezing of tissues was considered in calculation of protein synthesis rates (see below) according to Muramatsu et al. (1983) .

At d 23 and 29 of age, respectively, seven birds per treatment corresponding to their average group live weight were processed as described above in order to calculate k_s-related fractional tissue protein growth rates (k_g).

Viscosity measurements were carried out at d 23 of age. Seven birds per treatment representing their average group live weight were killed, and jejunum and ileum, as defined above, were quickly dissected. Digesta was stripped out, homogenized and placed on ice until further processed.

Preparation of tissue samples.

Tissue samples were finely ground in a freezer mill (Model 6700–230; SPEX CertiPrep, Metuchen, NJ) under liquid nitrogen. Powdered samples (~300 mg) were exactly weighed into precooled test tubes to which three small glass beads and 3 mL of ice-cold 0.25 mol/L perchloric acid were added. Samples were then mixed thoroughly and, after standing on ice for 10 min, centrifuged at 10,000 x g for 15 min at 4°C. The supernatant, which contained the free amino acids, was decanted and stored frozen (-20°C) until further analysis. The pellet was resuspended in 4 mL of 0.25 mol/L perchloric acid, centrifuged as above and the supernatant discarded. This washing procedure was repeated to ensure removal of all traces of free amino acids from the protein pellet, which were then dissolved in 8 mL of 0.3 M NaOH and incubated at 37°C for 1 h. From this digest, 0.5 mL was taken for further analysis of protein concentration. Next, 2 mL of ice-cold 1.5 mol/L perchloric acid was added and the tubes placed on ice for 20 min followed by centrifugation at 10,000 x g for 15 min at 4°C. The supernatant was transferred into test tubes and kept frozen for analysis of RNA-concentration. The pellet was washed twice with 4 mL of ice-cold 0.25 mol/L perchloric acid and finally incubated with 2 mL of 0.6 mol/L perchloric acid at 70°C for 1.5 h. The incubated sample was then placed on ice for 10 min before being centrifuged as described above. The supernatant was transferred to test tubes and kept frozen for subsequent DNA analysis. Finally, 6 mL of 11 mol/L HCl was added to the pellet and hydrolyzed for 24 h at 110°C. Sample (3 mL) was dried under a stream of nitrogen, washed with 1 mL of distilled water and dried down again for preparation for [¹⁵ N] phenylalanine analysis of protein bound phenylalanine.

Analysis of protein concentration was performed on day of sample preparation whereas RNA- and DNA-concentrations were analyzed later using the respective frozen supernatants.

Preparation of free amino acids for [¹⁵N] phenylalanine analysis.

The supernatant, containing the free amino acids, was thawed and loaded onto 1.2 mL of AG-50W-X8 cation exchange resin (H⁺ form, 100–200 mesh; Bio-Rad Laboratories GmbH, München, Germany) and washed with 4 mL of distilled water. Amino acids were eluted with 2 mL of 2 M NH₄OH followed by 1 mL of distilled water. The eluate was dried under a stream of nitrogen and then derivatized to yield the tertiary-butyldimethylsilyl (t-BDMS) derivative, as described by Calder and Smith (1988) . The free pool amino acid derivatives were analyzed on a gas chromatograph mass spectrometer (Shimadzu GC-17A attached to a Shimadzu QP-5000; Shimadzu Corporation, Kyoto, Japan). The peak area ratios for the m-57 fragment ions at m/z 336 and 337 of the phenylalanine-t-BDMS derivative were recorded in the selective ion monitoring mode and used for the calculation of the atom percentage excess of phenylalanine in the respective samples, based on the equation of Campbell (1974) .

Preparation of hydrolysates for [¹⁵N] phenylalanine analysis.

The dried hydrolysate was dissolved in 1 mL of 0.1 mol/L HCl, bound on 1.2 mL of the AG-50W-X8 cation exchange resin (H⁺ form), and washed with 4 mL of distilled water. Amino acids were eluted with 2 mL of 2 M NH₄OH followed by 1 mL of distilled water. The eluate was taken to dryness under a gentle stream of nitrogen at 60°C. The dried amino acids were converted to their t-BDMS derivatives, as described for the free amino acids. Phenylalanine enrichment was measured by GC-C-IRMS. The system used consists of a GC HP 6850 for separation of amino acids (fused silica column of 30 m length, i.d. of 0.32 mm and a film thickness of 1 µm, HP-5: cross-linked 5% PH ME silicone, He as carrier gas, injection of 1 µL sample in a splitless mode corresponding to ~4 nmol of phenylalanine). The sample then entered a preparation module (ORCHID, Europa Scientific Limited, Crewe, United Kingdom) for combustion of individual amino acids to nitrogen oxides, subsequently reduced to N₂, and for removal of water and CO₂ from the sample. The purified N₂ was then diverted to the 20–20 mass spectrometer (Europa Scientific Limited), and the [¹⁵ N] enrichment of the separated phenylalanine was calculated from the ratio of m/z ions 28 and 29 (GC-postprocessor; Europa Scientific Limited) using air nitrogen as standard.

Analysis of protein- RNA- and DNA-concentration in tissues.

Tissue composition was analyzed according to a modified Schmidt-Thannhauser method using subsamples obtained during sample preparation (see "Preparation of tissue sample" section). Protein concentration was analyzed in the 0.3 M NaOH digest of the sample according to Ohnishi and Barr (1978) using bovine serum albumin as standard. RNA concentration was determined in the supernatant obtained after treatment of the 0.3 M NaOH digest with 1.5 M perchloric acid using the cupric ion catalyzed orcinol method as described by Lin and Schjeide (1969) using purified yeast RNA as standard. Finally, the diphenylamine method was applied for analysis of DNA concentrations according to Burton (1956) using purified DNA-salt as standard in the supernatant centrifuged after incubation of the sample with 0.6 M perchloric acid at 70°C.

Viscosity measurements.

Digesta of jejunum and ileum was centrifuged at 15,000 x g for 15 min. Viscosity of supernatants was then measured at a rotary viscometer (Brookfield viscometer, model DV-II + LV; Brookfield Engineering Laboratories, Middleboro, MA) at 40°C.

Calculations and statistics.

The k_s (%/d) was calculated according to McNurlan et al. (1979) :

where APE_b is the [¹⁵ N] atom percentage excess (relative to natural abundance in samples from untreated chicks) of phenylalanine in protein, APE_f is the [¹⁵ N] atom percentage excess of free phenylalanine in tissues, assumed as the precursor pool, and t is time, expressed in days.

The k_g were calculated from the linear regression of time (d) on protein mass (mg) of individual tissues at d 23, 26 and 29 of age, respectively. The slope of the regression line was divided by the respective tissue protein mass at day of measurement of k_s (d 26 of age) and multiplied by 100 to obtain a k_g which is time-related to k_s.

Data were analyzed according to a complete two by two two-factorial design of ANOVA:

where y_ijk = k^th observation for fat type i and enzyme supplementation j, a_i = fat type (S, T), b_j = enzyme supplementation (‘-’ and ‘+’ xylanase supplementation), a_ixb_j = interactions between fat type and enzyme supplementation, e_ijk = error term.

Significant differences between the means were detected by the Tukey’s honestly significant difference test for unequal N.

	RESULTS

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Performance and intestinal viscosity.

Feed intake was significantly depressed in birds fed the unsupplemented tallow containing diet at d 23, 26 and 29 of age. Corresponding live weights were significantly influenced by fat type and enzyme supplementation (Table 2 ). Birds fed S containing diets were significantly heavier than T-fed chickens and xylanase addition increased weight more dramatically in T-fed birds than in S-fed birds (significant interaction between fat type and enzyme supplementation, Table 2 ).

Weight and protein mass of tissues.

Relative weights (per kg live weight) of the duodenum, jejunum and ileum were significantly affected by xylanase supplementation at all ages of the birds and by fat type in some instances (Table 3 ). This was due to the greatly enhanced weight of intestinal tissues of (T-)-fed birds at all ages and of (S-)-fed birds at d 23 of age. Xylanase supplementation significantly decreased intestinal weights especially in T-fed birds. The more pronounced decrease in intestinal weights caused by enzyme addition to the T containing diet resulted in the significant interactions between fat type and enzyme supplementation. Similarly, relative weights and amounts of protein of pancreas were greater in birds fed the unsupplemented diets at d 26.

Generally, RNA- and DNA-mass reflected the trends as described for relative tissue weights (Table 4 ). RNA- and DNA-mass of pancreas and intestinal tissues and RNA-mass of liver were higher in birds fed unsupplemented diets, especially in those fed the T-containing diet. The RNA to protein ratio and RNA to DNA ratio were also greater in these birds albeit not significantly. A disproportionate increase in RNA relative to protein mass on the one hand and in RNA relative to DNA on the other hand might have caused the increased ratios in (T-) fed birds. In addition, T feeding resulted in higher protein to DNA ratios in ileum, liver and pancreas.

APE of free tissue phenylalanine was 30.9 ± 1.5, 30.9 ± 1.9, 24.1 ± 4.2, 30.1 ± 2.2, 31.8 ± 1.1, 30.5 ± 1.0, and 33.9 ± 0.7 in duodenum, jejunum, jejunal mucosa cells, ileum, liver, pancreas and blood plasma, respectively. Values were not influenced by dietary treatments.

Tissue protein synthesis and protein growth

Feeding the unsupplemented T-containing diet resulted in significantly enhanced k_s in all intestinal segments, in jejunal mucosa cells and in the pancreas, whereas there were no differences between the remaining three treatments (Table 5 ). Liver-k_s was significantly increased after xylanase addition in both fat type diets. Intestinal- and pancreatic k_g reflected essentially the same relationships as described for k_s whereas in the liver an inverse relationship was observed since liver k_g significantly decreased after enzyme supplementation.

The differences in liver ASR between groups were significant when related to live weight. The ratio between ASR and RNA was not influenced by dietary treatments, although a trend for higher ratios was observed in enzyme-supplemented groups. The amount of protein synthesized per unit DNA was significantly and slightly (P = 0.134) increased after enzyme addition to the S- or T-containing diet, respectively.

	DISCUSSION

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Rye-based diets, either unsupplemented or supplemented with a xylanase containing enzyme preparation, were used in the present experiment to provoke large differences in intestinal viscosity. Soluble rye pentosans mediate their anti-nutritive effects in broilers by an increase in intestinal viscosity (Bedford and Classen 1992 ). Feeding of the unsupplemented and T-containing diet amplified the negative effects as indicated by the depressed feed intake, which was accompanied by a decrease in live weight of these birds. However, differences in feed intake between groups disappeared when feed intake was expressed on a metabolic live weight basis due to parallel decreases in feed intake and in live weight.

From the present results, it becomes clear that feeding of the unsupplemented T-containing diet greatly increased relative weight and protein mass of intestinal tissues including pancreatic tissue. The difference between k_s and k_g of secreting tissues such as the small intestine, pancreas and liver is a result of both the breakdown rate of constitutive tissue proteins but also the secreted proteins since the technique as applied in this experiment (10-min measurement period) measures this component as well (McNurlan et al. 1979 ). Consequently, k_s of small intestine, pancreas and liver represents a composite measure of protein synthesis for tissue maintenance and secretory protein output.

The fact that tissue weight, tissue protein mass, k_g and k_s in duodenum, jejunum and ileum were significantly increased in a highly viscous intestinal environment (caused by feeding an unsupplemented rye-based, T-containing diet) is in agreement with the findings of studies where effects of substances with gel-forming properties on mucosal cell turnover were investigated. Feeding guar gum and carboxymethyl cellulose was shown to increase the cell division rate in the small intestine, colon and cecum of rats which was accompanied by a reduced activity of some mucosal enzymes (Johnson et al. 1984 , Johnson and Gee 1986 ). In agreement with the findings for rats, the crypt cell proliferation rate of broilers was decreased on xylanase supplementation of a rye-based diet, a treatment which resulted in concomitant reduction in intestinal viscosity (Silva and Smithard 1997 ). Furthermore, Langhout et al. (1999) measured an increased number of goblet cells per 100 villus cells when high- or low-methylated pectin was added to a corn-based broiler diet. It was suggested that a higher density in goblet cells may result in an increased secretion of mucin. The increased k_s as observed in this experiment for T(-) birds may therefore be a result of increased protein secretion of mucosal cells.

Enzyme supplementation of the S-containing diet did not alter protein synthesis of small intestine to a great degree. This is of special interest because intestinal viscosity was more than twice in (S-)-fed birds compared to (S+)-fed birds, although it was significantly lower than that of the (T-)-birds. It might be concluded, therefore, that either intestinal viscosity in the presence of S oil does not stimulate intestinal protein synthesis, or that S protects the villi when compared with T-fed birds. Sagher et al. (1991) reported such protective effects in rats fed corn oil when compared to butter fat.

Small intestinal microbial composition and density are involved in interactions with mucosal metabolism and protein synthesis. Rolls et al. (1978) compared germ-free and conventional chicks and found that the presence of microflora promoted greater mitotic activity, faster migration and a more rapid turnover of epithelial cells in the intestinal mucosa. Moreover, protein synthesis in mucosal cells, in duodenum, jejunoileum and ceca of conventional chicks was found to be higher than in germ-free chicks (Muramatsu et al. 1983 , 1988). The altered intestinal microbial population or activity as shown by Dänicke et al. (1999a) in a related experiment might contribute to stimulation of intestinal protein synthesis in (T-)-fed birds.

Measurement of nucleic acids (Table 4) generally revealed a similar picture to protein synthesis measurements. RNA- and DNA-concentrations can be interpreted as indicators of number of ribosomes or intensity of protein synthesis and of rate of mitosis or hyperplasia, respectively. The increased k_s in (T-)- fed birds was based on both an increased number of cells or nuclei and an increased number of ribosomes combined with an increased protein synthetic capacity, the latter being described by the ratio of RNA to protein. Cell size can be described by the ratio of protein to DNA. It becomes clear that cell size remained unchanged in duodenum and jejunum, whereas an increase was observed in ileum of (T-)-fed birds.

When expressed per kg live weight, ASR in duodenum and jejunum of (T-)-fed birds was ~1.2 to 1.4 g per day higher than in the other groups. Translational efficiency or RNA-activity is characterized by relating the ASR to RNA, whereas the ratio of ASR to DNA indicates the DNA activity. From these figures it can be seen (Table 5) that DNA activity was greatly enhanced in (T-)-fed birds, whereas translational efficiency was comparable to the other groups, suggesting that greater concentrations of protein were synthesized per cell due to a higher cellular concentrations of RNA.

Pancreatic protein synthesis as measured by the methods applied in this experiment relates not only to constitutive protein synthesis but also to protein synthesis of both the endocrine and exocrine pancreas. Both synthesis and secretion of enzymes from the exocrine pancreas are backregulated by feed intake and nutrient presence and density in the intestine. An approximately twofold increase in flow of pancreatic juice was measured within 30 min after beginning of a meal in cockerels after 15-h feed withdrawal (Niess 1981 ). In periods of feed deprivatio, proteins accumulate in the pancreas, partially as a result of downregulation of secretion (Nir and Nitsan 1979 ). Such observations might also explain some part of the variation observed in this experiment (Table 5) , despite the fact that attempts were made to standardize feed intake through use of a 4-h fasting period. Pancreatic protein mass, k_g, k_s and nucleic acid concentrations were significantly increased in (T-)- fed birds which would indicate an hypertrophic pancreas which synthesized high amounts of secretory enzymes or proteins. Indeed Isaksson et al. (1983) found an increased lipase output in rats fed a pectin-containing diet. Total fat excretion in the feces was increased at the same time. Dänicke et al. (1999b) reported an increased pancreatic weight and an enhanced lipase activity (units per kg live weight) in birds fed an unsupplemented T-containing rye-based diet. It might be concluded therefore that the increased k_s as observed in this experiment in (T-)- fed birds might be due to an increased synthesis and secretion of lipase and other digestive enzymes.

Liver metabolism was also significantly influenced by dietary treatments but by different mechanisms (Table 5) . Whereas k_s was increased in enzyme supplemented groups, the opposite was the case for k_g. The xylanase-caused increase in k_s could simply reflect a higher nutrient and energy availability for synthesis of liver proteins to be exported since feed deprivation reduced liver k_s in broilers (Kita et al. 1996 ), but this effect seemed to be dependent on the preceding energy supply (Nieto et al. 1994 ). Moreover, protein supply was shown to modify liver k_s (Kita et al. 1996 ).

The main conclusion of this study is that effects of soluble nonstarch polysaccharides and of NSP-hydrolyzing enzymes may be explained partially by modifications in tissue protein metabolism of intestinal tract and that fat type is an additional nutritional determinant for these processes.

	FOOTNOTES

 
¹ Experiment was supported, in part, by Finnfeeds International, Marlborough, United Kingdom.

³ Abbreviations used: APE, [¹⁵N]-atom percentage excess; ASR, absolute rate of protein synthesis; GC-C-IRMS, gas chromatography-combustion-isotope ratio mass spectrometry; GC-MS, gas chromatography-mass spectrometry; k_s, k_g, fractional rates of tissue protein synthesis and growth, respectively; NSP, nonstarch-polysaccharides; (S-) or (T-), soybean oil- or beef tallow-fed birds, no xylanese in diet; (S+) or (T+), soybean oil- or beef tallow-fed birds, xylanese added to the diets; t-BDMS, tertiary butyldimethylsilyl.

Manuscript received June 17, 1999. Initial review completed August 10, 1999. Revision accepted November 29, 1999.

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