Chemical and in vivo evaluation of a brown midrib mutant of Zea mays. I. Fibre, lignin and amino acid composition and digestibility for sheep

Two types of corn (maize) were used which were genetically identical except for a mutant gene (bm₁) in one which reduced the type and amount of lignin produced compared with the normal plant (Tr). The Tr plant appeared to have only slightly lower digestibilities of dry matter and fibre for sheep even though the protein contents of the cornstalks, normally less in the Tr, were equalised by supplementation with soya bean protein. Comparing the acid-detergent method of fibre and lignin determination with an acid-pepsin method revealed that the former method gave values considerably lower than the latter. The alkali—lignin contents of the bm₁ were higher than the acid—detergent lignins which must under-estimate the true lignin content. The acid—pepsin method gave fibre and lignin concentrations which were more closely correlated with rumen digestion of the diets as shown by dry matter loss of the diets suspended into the rumen in nylon bags. This technique also revealed there was a significant increase in the rate of rumen digestion after 69 to 74 h of incubation. Digestion of lignin on the Tr diet could be explained by loss of ferulic and p-coumaric acids. The reduced lignin and p-coumaric acid content of the bm corn was associated in young plants with a low phenylalanine content but normal tyrosine content suggesting that the genetic block in lignin synthesis in this plant may occur between prephenic acid and phenylalanine.     

A comparison of some chemical and physical properties of alkali lignins from grass and lucerne hays before and after digestion by sheep

The chemical and physical properties of alkali lignins from a grass hay and a lucerne hay were studied, before and after digestion by sheep. A greater proportion of the total grass lignins was dissolved by alkali than with the lucerne lignins but digestion increased this proportion with both species. Of the lignin dissolved by alkali, less of it was precipitated on acidification when faeces (50–70%) were compared with the undigested hays (86–92%). Extraction of lignins with boiling alkali produced a lignin with the smallest amount of amino acids but considerably altered the chemical structure as revealed by nuclear magnetic resonance (n.m.r.). Trypsin treatment of a cooler alkali extract was less effective in reducing amino acid contamination but did not alter the structure. Compared with the grass alkali lignins, the lucerne lignins had more total but fewer aromatic protons, fewer groups reacting with N-2,6-trichloro-p-benzoquinone imine, ionisable hydroxyl groups and fewer methoxyl groups. On Sephadex G-200 gel chromatography, three peaks were apparent with the lucerne, but only two with grass lignins. Both plant lignins showed large amounts of highly shielded n.m.r. protons but there were more with lucerne. Infrared spectra suggested that they were probably due to methyl groups. Digestion had very little effect on the n.m.r., ultraviolet or infrared spectra or the molecular weight distribution of the isolated lignins. It may have reduced the numbers of groups, reacting with N-2,6-trichloro-p-benzoquinone imine with the grass and two maize samples examined, but appeared to have the opposite effect with the lucerne.     

Comparative characterisation of dioxane-soluble lignins released by ball milling and by sheep digestion from forage grasses

Faecal soluble lignin (FSL), extracted from the faeces of sheep that received orchardgrass and timothy, were compared with 90% dioxane-soluble lignins released by ball milling (MHL) and by subsequent enzymatic hydrolysis (EHL) from the same grasses. FSL contained much less carbohydrate and esterified p-coumaric and ferulic acids than MHL and EHL. Although no considerable differences were found in the yields of vanillin and syringaldehyde produced by nitrobenzene oxidation, the molar ratio of syringaldehyde to vanillin was higher for FSL than for MHL and EHL. Such differences in chemical properties among the lignin preparations were confirmed by ¹³C-NMR spectroscopy. FSL had a lower molecular size compared with MHL and EHL. Results indicated that syringyl-rich lignin fragments with less phenolic acid esters, probably depolymerisation products of the grass lignins, were released by digestion in sheep from the forage grasses.     

14C labelling of lignins of normal and bm3 maizes for fermentation studies

[U-¹⁴C] phenylalanine (phe*) and [O¹⁴CH₃] sinapic acid (sin*) were infused into the cut ends of normal and bm3 maizes (anthesis stage) under or above the last node or at mid-internode, with or without the leaf, in light or in darkness. Radioactivity was measured in the organs, and in phenolic constituents of the cell wall and saponified residues of the bases and tops of the apical inter-node. In both maize genotype labelled under the node the radioactivity was distributed more evenly in the organs with sin* than with phe*. Infusion above the node and at mid-internode greatly increased radioactivity in the bases and tops, respectively. Removal of the leaf only slightly increased the radioactivity, mainly in the bases, and no clear-cut effect of darkness was observed. Phe* labelled the phenolic acids and the three lignin units, but the syringyl units of bm3 maize were only slightly labelled. Sin* specifically labelled the syringyl units, which represented the least condensed fraction of lignins. Both the native and labelled lignins were highly alkali soluble. There were differences in lignin biogenesis between the bases and tops, and between normal and bm3 maizes. The newly formed lignins were slightly different from the native lignins but had similar types of heterogeneity, with variations in the internode and between genotypes similar to those in native lignins. Provided due allowance is made for the distinguishing characteristics of newly formed lignins, the [¹⁴C-lignin] cell walls, which are strongly labelled on complementary structures, seem suitable model substrates for fermentation studies.     

Severe inhibition of maize wall degradation by synthetic lignins formed with coniferaldehyde

Although the enzymatic or ruminal degradability of plants deficient in cinnamyl alcohol dehydrogenase (CAD) is often greater than their normal counterparts, factors responsible for these degradability differences have not been identified. Since lignins in CAD deficient plants often contain elevated concentrations of aldehydes, we used a cell-wall model system to evaluate what effect aldehyde-containing lignins have on the hydrolysis of cell walls by fungal enzymes. Varying ratios of coniferaldehyde and coniferyl alcohol were polymerised into non-lignified primary walls of maize (Zea mays L) by wall-bound peroxidase and exogenously supplied H₂O₂. Coniferaldehyde lignins formed fewer cross-linked structures with other wall components, but they were much more inhibitory to cell wall degradation than lignins formed with coniferyl alcohol. This suggests that the improved degradability of CAD deficient plants is not related to the incorporation of p-hydroxycinnamaldehyde units into lignin. Degradability differences were diminished if enzyme loadings were increased and if hydrophobic aldehyde groups in lignins were reduced to their corresponding alcohols by ethanolic sodium borohydride. © 1998 Society of Chemical Industry.     

Biological variability in lignification of maize: Expression of the brown midrib bm2 mutation

The cell wall phenolic components in the internodes of three maize genotypes, namely normal, bm2 and bm3 maize, were determined. The bm2 and bm3 brown midrib mutations lowered the lignin content of the bottom, middle and top internodes to a similar extent. However, unlike bm3, the bm2 trait did not induce a sharp reduction of the level of ester-bound p-coumaric acid in maize internodes. The other main alkali labile phenolic acid, ferulic acid, reached similar levels in the three genotypes. The main difference between bm2 and bm3 mutations occurred in the alkyl aryl ether linked structures of the lignin component. In contrast to bm3 lignins, which are characterised by a low syringyl content, the bm2 lignin had a lower content of guaiacyl units than lignin of normal maize internode. Consequently, the syringyl/guaiacyl molar ratio of bm2 lignin gave higher values (2.7–3.2) than those from either normal (0.9–1.5) or bm3 lignins (0.3). The alkali solubility of lignin was also compared between the three genotypes. Incorporation of the bm3 trait in maize led to a high recovery of alkali soluble lignin whereas the bm2 lignin had a similar solubility to the normal one in 2 M NaOH. The monomeric composition of the alkali soluble lignins was consistent with the non-condensed structures of the in-situ polymer. Although the bm3 and bm2 mutations had different effects on lignification, the modification of the cell wall phenolic level was also found in the bm2 maize stem as previously studied.     

Determination of lignin in herbaceous plants by an improved acetyl bromide procedure

A modified acetyl bromide procedure is proposed for the spectrophotometric determination of lignin in herbaceous plants. The digestion with 25 % acetyl bromide (AcBr) in acetic acid at 70°C is improved by including 4 % perchloric acid in the digesting solutions. This allows for more rapid, complete digestion and the use of coarser samples. On the basis of infrared spectra and nitrobenzene oxidation data for milled sample lignins, the value of 20.0 g^?1 litre cm^?1 for the specific absorption coeficient of AcBr-treated lignins of herbaceous plant samples gave lignin values which were consistent with nitrobenzene oxidation data, but were higher than those obtained by the acid detergent lignin method and the earlier AcBr method proposed for grasses. The lower lignin values obtained in the latter methods are considered to be due to partial lignin solubilisation in the sulphuric acid digestion.     

Phenolic components and degradability of the cell walls of the brown midrib mutant,bm3, of Zea mays

Cell walls separated from the leaf blade, leaf sheath and stem of the brown midrib mutant, bm₃, of Zea mays were more degradable by a commercial cellulase than the corresponding part of the isogenic normal inbred cultivar (Tr). The walls of each part of the mutant when compared with the corresponding part of the normal cultivar contained less lignin and bound phenolic components released by treatment with NaOH. The major phenolic components detected were trans-p-coumaric and trans-ferulic acids together with small amounts of their cis isomers and diferulic acid. Cell walls of stem of the mutant contained a total of 17.3 mg g^?1 of these bound acids compared with 9.8 mg g^?1 for leaf sheath and 3.5 mg g^?1 for leaf blade: there was more than twice as much p-coumaric acid in cell walls of stem as in those of leaf sheath and more than seven times as much as in those of leaf blade. When cell walls of the stem from the mutant or the normal cultivar were treated with NaOH their degradability by cellulase was highly correlated with the amounts of phenolic components released by the alkali.     

Fractionation and characterization of ball‐milled and enzyme lignins from abaca fibre

An Erratum has been published for this article in Journal of the Science of Food and Agriculture 79(15) 1999, 2122. Ball‐milled and enzyme lignins were produced from abaca fibre via ball milling for 6 days followed by cellulase treatment for 3 days. The crude lignin preparations were fractionated into milled lignin (ML), enzyme lignin (EL), hemicellulose‐rich milled lignin (HRML), and lignin‐rich enzyme lignin (LREL) fractions using a two‐step precipitation method instead of a traditional ether precipitation procedure. The yield and chemical composition of the resulting lignin samples are reported. The ML and EL fractions contained low amounts of associated neutral sugars (2.0–3.3%) and uronic acids (1.4–1.5%), and showed relatively low average molecular weights (2500–2660), while the LREL and HRML fractions contained large amounts of bound polysaccharides (35.6–38.3%), and showed high molecular weights (8800–25000). The four lignin fractions are composed of a large proportion of syringyl units with fewer guaiacyl and p‐hydroxyphenyl units. The ML is mainly composed of β–O–4 ether bonds between the lignin structural units. The less common β–β, β–5 and 5–5′ carbon–carbon linkages are also present in the lignin molecules. It was found that uronic acids and 41–63% of p‐coumaric acids are esterified to lignin in the three lignin‐rich fractions of ML, EL and LREL. This level increased to over 90% in the hemicellulose‐rich fraction of HRML. For ferulic acids, 92–97% were found to be etherified to lignin in the three lignin‐rich fractions of ML, EL and LREL, while in the hemicellulose‐rich fraction of HRML this reduced to only 13%, suggesting that a majority of the ferulic acids are esterified to hemicelluloses or lignin in this fraction. © 1999 Society of Chemical Industry     

Intake and Digestibility of Brown-Midrib Corn Silage by Lactating Dairy Cows,

Two short trials in 2 yr evaluated brown midrib-3 (bm3) mutant (low lignin) corn silage for Holstein cows in early lactation. In trial 1, five Holstein cows per group (bm3 or normal genetic counterpart) were fed individually a ration of corn silage and grain (60:40, dry matter) from wk 2 through wk 8 postcalving. Cows fed the bm3 ration consumed 19% more digestible dry matter and energy as a percent of body weight than cows fed the normal ration. Digestibilities of most components were 2 to 4 percentage units higher for the bm3 ration. Milk yield was not different between rations, but cows fed bm3 lost less body weight. In trial 2, five Holstein cows per group were fed individually a ration of either bm3 or normal silage supplemented with a concentrate mix at 85:15 silage to concentrate. The concentrate mix was formulated to meet needs for protein, minerals, and vitamins. Cows were on trial from wk 6 through wk 13 postcalving. All measures of daily intake of dry matter were greater for the bm3 ration while digestibilities of some components were reduced slightly. Digestible energy intake was 27% greater for the bm3 ration. Milk and milk component yields did not differ, but cows fed the bm3 ration gained more body weight. Total and individual ruminal volatile fatty acids were higher and pH lower with time after feeding for the bm3 ration.     

Fungal pretreatment of wheat straw: Effects on the biodegradability of cell walls,structural polysaccharides,lignin and phenolic acids by rumen microorganisms

Disappearance of cell wall components of untreated straw and straw treated with the ligninolytic white-rot fungi Phanerochaete chrysosporium, Dichomitus squalens and Cyathus stercoreus were determined during the course of rumen digestion of samples in nylon bags. The first fungus degraded hemicelluloses and cellulose non-selectively, adversely affecting the digestion rate of crude cell walls. Dichomitus squalens and C. stercoreus preferentially degraded hemicelluloses and lignin, affording cell wall degradation rates 1.5 times higher than in native straw. Furthermore, the extent of cell wall digestion was also significantly enhanced. Both strains improved the extent of cellulose digestion, whereas the potentially degradable xylan fraction remained unchanged. Polysaccharide digestion rates were influenced in different ways depending on the strain tested: straw degraded by C. stercoreus showed an increase in cellulose digestion rate by 50%, whereas residual arabinose units were slowly degraded. Xylan was degraded 1.8 times faster in straw decayed' by D. squalens, while cellulose digestion remained unchanged. Phanerochaete chrysosporium depressed both xylan and cellulose digestion rates. Fungal-treated lignins were solubilised in the rumen faster than in untreated straw, whereas only treatment by C. stercoreus resulted in higher lignin losses. Esterified phenolic acids were extensively degraded by all three fungi. Residual ferulic and p-coumaric acids accumulated during rumen digestion, although only the former decreased in the original straw.     

Characterisation of Lignin from CAD and OMT Deficient Bm Mutants of Maize

Internodes of the maize cell line W401 and bm₁ and bm₃ mutants expressed in W401 were harvested 5 days after anthesis (A5) and at silage (S) stage. The normal maize had a higher total phenolic (TP) content (80·5–90·5 g kg^-1 cell wall DM) than both bm₁ and bm₃ mutants (74·4–86·4 and 66·0– 84·2 g kg^-1 cell wall DM, respectively). TP were inversely related to cellulase digestibility with values of 85·4–91·5, 89·3–92·1 and 91·3–94·1% for normal, bm₁ and bm₃. Marked differences in p-coumaric acid concentrations were found ranging from 20·9 to 26·3 g kg^-1 cell wall DM for normal, 14·9 to 15·3 g kg^-1 for bm₁ to 10·1 to 14·4 g kg^-1 for bm₃. The ferulate pattern was entirely different with the bm₁ genotype providing the lowest total (9·1–10·7 g kg^-1) and etherified (1·9–2·3 g kg^-1) values. Although the bm₃ contained more total ferulate (11·5–13·1 vs 10·9–11·7 g kg^-1), the normal variety had a significantly greater amount of etherified ferulate (2·8–3·4 vs 3·2–4.1 g kg^-1) implying a greater extent of cross-linking between wall polymers. Recovery of guaiacyl and syringyl residues was greatest in the normal maize with the bm₁ occupying the middle position between the two extremes. Calculated S: G ratios from 4 M NaOH digestion and NMR were in good agreement with the normal line giving the highest ratio, bm₁ intermediate and bm₃ the lowest. Colorimetric analysis revealed a large increase in the aldehyde content of the in situ bm₁ lignin compared to normal and bm₃ genotypes although NMR failed to reveal significant numbers of aldehydic resonances. © 1997 SCI.     

The composition and chemiluminescence of acidolysis lignins from native Chilean hardwoods

Nothofagus obliqua (roble blanco), Nothofagus glauca (roble colorado), Lithraea Caustica (litre), Quillaja saponaria (quillay) and Cryptocarya alba (peumo), classified as hardwood species, were subjected to chemical and spectroscopic analysis. A high extinction coefficient at 280 nm correlates with the high free phenolic group content of the lignins of these species. Nothofagus glauca exhibited a high methoxyl content (19.3%) and the lower phenolic content (1.38%). The chemiluminescent behaviour of these lignins was found to be directly proportional to phenolic groups content and inversely proportional to the methoxyl content.

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Pyrolytic and hydrogenolytic degradation studies on lignocellulosics,pulps and lignins

Wood and MWL's from beech, spruce, bamboo as well as teak wood and teak-HCl-lignin were subjected to analytical pyrolysis (Py), using the off-line approach, and to hydrogenolysis (Hy). Gas chromatography-mass spectrometry (GCMS) was used for product identification and assignment as derivatives from 4-hydroxy-phenyl-propane, guaiacylpropane and syringylpropane basic units followed by capillary gas chromatography (GC) for quantitative determination of the phenolics. The results are compared with those of nitrobenzene oxidation and quantitative FTIR-spectroscopy. Py-GC and Hy-GC experiments were also used for the characterization of residual lignins in kraft and alkaline-sulphite-anthraquinone-methanol (ASAM) pulps from beech, pine and sugar cane bagasse. Analytical pyrolysis of biomass, both in on-line and off-line approach, is well suitable for lignin classification even without previous lignin isolation. Hydrogenolysis is more recommendable for the characterization of residual lignins in pulps.     

A comparative study of hard and softwood lignins alterations during treatment in dioxane-water-HCl agent

The objective of the presented contribution was a study of the lignin alterations occurring in the process of the acidolytic delignification of chosen representatives of soft and hard wood species (maple and spruce) in dioxane-water-HCl mixture. All lignin samples were characterised by yield or recovery, molecular weights, as well as by GLC data of corresponding nitrobenzene oxidation products. In order to obtain more information about condensation and degradative reactions of lignin under conditions of acidolytic treatment a simulation of the process of lignin isolation was performed, too. Samples of dioxane lignins obtained after 90 min. of isolation at 82.5 and 98 °C were therefore recooked during 360 min. under identical conditions as applied in the lignin isolations. Concentrations of lignin in the reaction mixture were 1 and 10% thus approximately corresponding the lignin concentration inside and outside of the wood meal during the lignin isolation. The obtained experimental data point out the course of both degradative and condensation reactions. Maple wood lignin undergo deeper degradation than those of spruce under the same reaction conditions. The decreased yields of lignin oxidation products with the time of acidolytic treatment and after recooking hint at the formation of new linkages among building units of lignin thus modifying its macromolecules. The intensity of occurring alterations of examined lignins is not only dependent of time and temperature, but also of the concentration of lignin.     

Roles of structural phenylpropanoids in forage cell wall digestion

Phenolic constituents (lignins and phenolic acids) and carbohydrates are assembled in a tight architecture which differs according to the plant species. During cell wall digestion, the hydrolysis kinetics differ between carbohydrates and seem to depend chiefly on the content and organisation of tissue phenolics. Among the phenylpropanoids, ferulic acid is released more quickly than p-coumaric acid. Lignins remain largely in the cell walls. They also undergo transformations, chiefly solubilisation as lignin-carbohydrate complexes. The limiting effect of lignins on cell wall degradation increases with increasing content. However, their effect on degradation might also depend on qualitative factors such as lignin structure and polymer organisation in walls and tissues. When various grasses (normal and selected genotypes), or grasses and legumes are compared, correlations between certain factors such as lignin uncondensed fraction, syringyl units or phenolic acids contents and cell wall degradation emerge but not clear causal relationship has been shown. Nonetheless, other structural characteristics, related to the alkali reactivity of lignins, seem to have a stronger influence on cell wall degradation. Phenylpropanoids seem to act mainly as a physical and (bio)chemical barrier to the action of the microbial enzymes. In addition, their reactivity as phenolic compounds and their hydrophobicity seem to play a role. Digestion is not limited only by phenolics. The factors that limit glycanolysis—the accessibility, crystallinity and capillary structure of cellulose and the branching of hemicelluloses—seem to have little or no effect on cell wall degradation in vivo. In contrast, other antiquality substances (tannins, cutin and silica), plant antomy, environmental factors, factors modulating microbial growth and animal physiology influence cell wall utilisation. Future research in this field should focus on the effects of phenolic structure and of cell wall and tissue organisation on carbohydrate degradation.     

Lignin accumulation and biosynthetic enzyme activities in relation to postharvest firmness of fresh waxy corn

Lignification and changes in the enzymes involved in lignin biosynthesis were investigated in relation to the reduced tenderness occurring during the postharvest storage of fresh waxy corn at 20 °C. The firmness of the corn and its lignin and polyphenol contents increased as storage time increased. The lignin content was significantly correlated (p < .05) with the polyphenol content and firmness. Phenylalanine ammonia‐lyase was not the rate‐limiting enzyme in polyphenol biosynthesis, whereas the cinnamate 4‐hydroxylase activity was significantly correlated (p < .05) with the polyphenol content. The activity of coenzyme A ligase (4CL) had a limited effect on the polyphenol content. Peroxidase was significantly correlated (p < .05) with the lignin content, while no significant (p > .05) effect of polyphenol oxidase or cinnamyl alcohol dehydrogenase (CAD) on the lignin content was observed.

Practical applications

Tenderness is a main factor in sensory quality of fresh waxy corn. Secondary cell wall lignifications induced by phenylpropanoid pathway is the main reason of tenderness decline for postharvest fresh waxy corn. But little information of lignin accumulation of fresh waxy corn is obtained from the present studies. Analyzing the changes of lignin and related precursor materials, enzyme activities will help to comprehensively understand the regulatory mechanism of postharvest lignification of fresh waxy corn, which can provide suitable methods to delay postharvest lignin accumulation, and maintain the eating quality of fresh waxy corn.     

Ultra-high pressure treatment effects on polysaccharides and lignins of longan fruit pericarp

Longan fruit pericarp was subjected to ultra-high pressure treatment. The yields of water-soluble polysaccharides, alkali-soluble polysaccharides and cellulose were comparatively analysed before and after ultra-high pressure treatment. A negative relationship was observed between pressure and water-soluble polysaccharide yield. The lowest yield (6.4 ± 0.6 mg/g) was obtained at 500 MPa. No significant differences (P > 0.05) in alkali-soluble polysaccharide and cellulose yields was found between the ultra-high pressure-treated and non-treated samples (control). Furthermore, a similar phenomena was observed for cellulose. The degrees of hydrolysis (DH) of control and 500 MPa-treated cellulose were 26.6% and 29.4%, respectively, and there was a significant difference (P < 0.05) between them. The degradation and oxidation of lignins were analysed using high performance liquid chromatography, and four main peaks appeared. A comparative profile suggested that ultra-high pressure treatment could not result in a change in the lignin composition.     

Lignin and Hydroxycinnamic Acids in Walls of Brown Midrib Mutants of Sorghum,Pearl Millet and Maize Stems

The characteristics of walls from stems of brown-midrib ( bmr ) mutants from Sorghum bicolor (L) Moench bmr6 and bmr18 (watery- to milky-grain stage), Pennisetum americanum (L) Leeke KS81-1089 (soft-dough stage) and Zea mays L bm3 (early-dent stage) with respect to the types of linkages of hydroxycinnamic acids to wall polymers and to structural features of their lignins were investigated. The lignin content of all mutants, determined using the acid detergent lignin procedure, was significantly lower than that of their normal counterparts. There was, however, no significant differences in total lignin contents between bmr and normal lines as determined by the acetyl bromide procedure or the sum of the acid-insoluble (Klason) lignin and acid-soluble lignin. It is suggested that this behaviour could be explained if bmr mutants are characterised by higher amounts of lignin with a lower degree of polymerisation than normal lines. The lowered S/V ratio and lowered total yield of alkaline nitrobenzene oxidation products in lignin from bmr mutants was confirmed. No etherified p -coumaric acid was found in any sample tested, except the normal line of pearl millet. The concentration of etherified ferulic acid, which is probably involved in ester-ether bridges between lignin and polysaccharides, was lower in bmr mutants than in the normal plants. The low content of ferulic acid bridges in bmr mutants may contribute to the elevated digestibilities of their stems.     

Influence of nonprotein nitrogen and protein of low rumen degradability on nitrogen flow and utilization in lactating dairy cows

Four lactating cows, each fitted with a rumen cannula and duodenal and ileal t-cannulae, were used to measure flow and digestion of nitrogenous compounds in the digestive tract. Dietary dry matter contained 17% crude protein and 50:50 forage:concentrate. Treatments were: 1) corn silage-soybean meal; 2) corn silage-heated soybean meal; 3) ammonia-treated corn silage-soybean meal; and 4) ammonia-treated corn silage-heated soybean meal. Flow of organic matter to the duodenum was overestimated when lanthanum or chromium ethylene-diaminetetraacetate was used as an indigestible marker. This resulted in low estimates of ruminal digestion of organic matter and high estimates of nitrogen flow to the duodenum. However, calculations using lanthanum or lignin as markers yielded similar organic matter flow to ileum and feces. With acid-detergent lignin as a marker, estimates of dietary nitrogen degraded in the rumen were: corn silage-heated soybean meal, 55.0% ammonia-treated corn silage-heated soybean meal, 58.8%; ammonia-treated corn silage-soybean meal, 63.3%; and corn silage-soybean meal, 66.0%. Digestion in the small intestine of nonammonia nitrogen was equal for all treatments suggesting that availability of heated soybean meal in the intestine was not different, although ruminal degradability tended to be lower. Feeding diets containing nonprotein nitrogen did not decrease available nitrogen at the duodenum.