Alfalfa Cell Wall Research Articles

Alfalfa Stem Cell Wall Digestibility: Current Knowledge and Future Research Directions

Alfalfa (Medicago sativa L.) is considered as the most important forage legume with high biomass yield and nutritional quality for ruminants. The alfalfa leaf cell walls are highly digestible, but stem cell walls of alfalfa are not readily digestible. The cell wall component of alfalfa has a large source of dietary energy, but ruminant animals can digest less than half of this component due to the presence of high lignin content. The main goal of this review is to make a summary of existing knowledge of alfalfa cell wall thickening and lignification patterns and suggest future directions for improving alfalfa stem cell wall digestibility. We describe alfalfa cell wall biochemistry, alfalfa stem morphology, stem tissue degradation, and existing methods to improve alfalfa digestibility, and discuss the potential future strategies for improving alfalfa cell wall digestibility. Information on these will help alfalfa breeders and producers identify superior alfalfa cultivars with improved stem cell wall digestibility. Concentrating future efforts on the selection and identification of traits and associated genes that affect cell wall digestibility could improve alfalfa cell wall digestibility.

Uptake, translocation and metabolism of di-n-butyl phthalate in alfalfa (Medicago sativa)

Uptake and metabolism by plants are important biotransformation processes of organic pollutants in ecosystems. However, very limited information is currently available on the metabolism of phthalic acid esters (PAEs) in plants. In this study, alfalfa, highly efficient in phytoremediation of PAE contaminated soil, was chosen as the model to understand the fate of di-n-butyl phthalate (DnBP) in remediation plant. The results of hydroponic experiments indicated that DnBP accumulated mainly in alfalfa roots and adsorption to root epidermis might be the primary uptake mechanism. A large proportion of DnBP was subjected to apparent metabolism. De-esterification could be specified to be the predominant metabolism pathway. Mono-n-butyl phthalate (MnBP) and phthalic acid (PA) were detected as DnBP metabolites in all alfalfa roots and shoots throughout the entire exposure period. Around >90% of MnBP were distributed in cell soluble components and organelles, and MnBP gradually transferred from organelles and cell walls to soluble components as the exposure time extended. Similar to MnBP, PA located mainly in soluble components and organelles as well, while no PA existed in alfalfa cell walls. Exposure to DnBP ultimately resulted in the coexistence of DnBP and MnBP for a long term in interior plants, raising concerns on their combined potential toxicity on plant health or even ecosystem.

Didehydrophenylalanine, an abundant modification in the beta subunit of plant polygalacturonases.

The structure and the activity of proteins are often regulated by transient or stable post- translational modifications (PTM). Different from well-known, abundant modifications such as phosphorylation and glycosylation some modifications are limited to one or a few proteins across a broad range of related species. Although few examples of the latter type are known, the evolutionary conservation of these modifications and the enzymes responsible for their synthesis suggest an important physiological role. Here, the first observation of a new, fold-directing PTM is described. During the analysis of alfalfa cell wall proteins a -2Da mass shift was observed on phenylalanine residues in the repeated tetrapeptide FxxY of the beta-subunit of polygalacturonase. This modular protein is known to be involved in developmental and stress-responsive processes. The presence of this modification was confirmed using in-house and external datasets acquired by different commonly used techniques in proteome studies. Based on these analyses it was found that all identified phenylalanine residues in the sequence FxxY of this protein were modified to α,β-didehydro-Phe (ΔPhe). Besides showing the reproducible identification of ΔPhe in different species arguments that substantiate the fold-determining role of ΔPhe are given.

Expression analysis of stem prickle formation in blackberry using cDNA-AFLP

To understand the molecular mechanism of prickle formation in blackberry, differential expression profile of cDNA between 'Boysenberry' blackberry and its thin-thorn mutant were analysed by using cDNA-AFLP. In total, 256 primer pairs were used for the cDNA-AFLP amplification to produce 45 differentially expressed fragments. The putative function of the sequenced fragments was determined using BLAST analysis. Putative functions included metabolism, signal transduction, cell skeleton and composition, transcriptional regulation and adversity defence. A 109 bp fragment was found to have an 88% sequence homology with a strawberry TRY transcription factor, which inhibits trichome development in Arabidopsis. A 124 bp fragment had a 75% homology with alfalfa cell wall hydrolase gene. This fragment was named Ruh and had higher expression in shoot apical meristem (SAM), middle and upper section of stem of thin-thorn mutant. This implied that the prickle formation was an intricate system and the sense of expression pattern of cell wall-associated hydrolase homologous gene Ruh in the thin-thorn mutant needs further investigation.

Expression of a fungal ferulic acid esterase in alfalfa modifies cell wall digestibility

BackgroundAlfalfa (Medicago sativa) is an important forage crop in North America owing to its high biomass production, perennial nature and ability to fix nitrogen. Feruloyl esterase (EC 3.1.1.73) hydrolyzes ester linkages in plant cell walls and has the potential to further improve alfalfa as biomass for biofuel production.ResultsIn this study, faeB [GenBank:AJ309807] was synthesized at GenScript and sub-cloned into a novel pEACH vector containing different signaling peptides to target type B ferulic acid esterase (FAEB) proteins to the apoplast, chloroplast, endoplasmic reticulum and vacuole. Four constructs harboring faeB were transiently expressed in Nicotiana leaves, with FAEB accumulating at high levels in all target sites, except chloroplast. Stable transformed lines of alfalfa were subsequently obtained using Agrobacterium tumefaciens (LBA4404). Out of 136 transgenic plants regenerated, 18 independent lines exhibited FAEB activity. Subsequent in vitro digestibility and Fourier transformed infrared spectroscopy (FTIR) analysis of FAEB-expressing lines showed that they possessed modified cell wall morphology and composition with a reduction in ester linkages and elevated lignin content. Consequently, they were more recalcitrant to digestion by mixed ruminal microorganisms. Interestingly, delignification by alkaline peroxide treatment followed by exposure to a commercial cellulase mixture resulted in higher glucose release from transgenic lines as compared to the control line.ConclusionModifying cell wall crosslinking has the potential to lower recalcitrance of holocellulose, but also exhibited unintended consequences on alfalfa cell wall digestibility due to elevated lignin content. The combination of efficient delignification treatment (alkaline peroxide) and transgenic esterase activity complement each other towards efficient and effective digestion of transgenic lines.

Changes in alfalfa cell wall structure during vegetation

The investigation was done on 141 samples of one alfalfa cultivar, collected from the same location during the first three growth cycles: spring growth, the first and the second regrowth. Within each growth cycle, sampling was done during the whole growing period, commencing when plant height was below 150 mm and continuing until plants were bearing ripe seeds. On all collected samples the following cell wall characteristics were determined: neutral detergent fibre (NDF), acid detergent fibre (ADF), acid detergent lignin (ADL), neutral detergent insoluble crude protein (NDICP), acid detergent insoluble crude protein (ADICP). Cellulose and hemicellulose were detected on the base of the mentioned chemical parameters. Significantly lower (p<0.01) content of aNDF, ADF, ADL, ADICP and cellulose is found in the second regrowth, while there were no significant differences between the other two growth cycles. Except in NDICP and ADICP, the increase in all accompanying components of the cell wall was observed, and expressed in average daily changes. There was no consistent trend in NDICP and ADICP. During the spring growth from late bud to full-bloom stage the ?plateau? was observed. The plateau was represented as almost constant content of aNDF, ADF, ADL and cellulose. The correlations between all components of the cell wall were shown. The equation aNDF = 36.713 + 1.181 ? ADF is recommended for conversion of ADF into aNDF in alfalfa.

Effects of nitrate adaptation by rumen inocula donors and substrate fiber proportion on in vitro nitrate disappearance, methanogenesis, and rumen fermentation acid

A study was conducted to evaluate the main effects of dietary nitrate adaptation by cattle and alfalfa cell wall to starch ratio in in vitro substrates on nitrate disappearance and nitrite and volatile fatty acid (VFA) concentrations, as well as hydrogen (H2) and methane (CH4) accumulations. Rumen fluid from steers fed diets containing urea or nitrate was added into in vitro incubations containing sodium nitrate as the sole nitrogen source and 20 cell wall : 80 starch or 80 cell wall : 20 starch as the carbohydrate source. The results showed that during 24 h incubation, rumen fluid inoculums from steers adapted to dietary nitrate resulted in more rapid nitrate disappearance by 6 h of incubation (P < 0.01), no significant effect on nitrite concentration and diminished CH4 accumulation (P < 0.05). Cell wall to starch ratio did not affect nitrate disappearance, CH4 accumulation and total VFA concentration. The higher cell wall ratio had the lower total gas production and H2 concentration (P < 0.05). Ammonia-N (NH3-N) concentration increased because of adaptation of donors to nitrate feeding (P < 0.05). Nitrate adaptation did not alter total VFA concentration, but increased acetate, and decreased propionate and butyrate molar proportions (P < 0.01).

Effect of fibrolytic enzymes and incubation pH on in vitro degradation of NDF extracts of alfalfa and orchardgrass

Two in vitro assays (one with alfalfa and the other with orchardgrass) were conducted to evaluate the effects of a fibrolytic enzyme preparation (enzyme) and initial incubation pH (5.6, 6.2 and 6.8) on the degradation of NDF extracts.The enzyme increased (P≤0.05) degradation of alfalfa NDF: 1) at 6 h with pH 6.2 and 6.8; 2) at 9 and 12h with pH 5.6 and 6.2; 3) at 24h with all pH values; 4) at 48h with pH 5.6 and 6.8; 5) at 72h with pH 6.2 and 6.8. Alfalfa NDF degradation was changed (P≤0.05) by the enzyme with increasing pH, as follows: 1) a linear increase at 3h; 2) a linear decrease at 6 and 12h; 3) a quadratic effect at 24, 48, and 72h, and the highest value was observed with pH 6.2. Further effects (P≤0.05) of the enzyme on the alfalfa cell wall were the following: 1) with pH 5.6, the undegradable NDF fraction was reduced and the degradation rate was increased; 2) with pH 6.2, the lag phase was decreased; 3) as pH increased there was a quadratic effect on the lag phase, and the lowest value was found at pH 6.2. For orchardgrass the effects (P≤0.05) of the enzyme were as follows: 1) an increased NDF degradation at 24 and 48h; 2) a decreased linear degradation of NDF as pH increased, at 3, 6, and 9h. Scanning electron micrographs of the alfalfa cell wall showed that parenchyma cells appeared broken and the vascular bundles exposed after incubation with the enzyme mixture at pH 6.2It may be concluded that a fibrolytic enzyme preparation at pH 6.2 enhanced in vitro degradation of alfalfa NDF.

Divergent phenotypic selection for alfalfa cell wall fractions and indirect response in digestibility

An alfalfa (Medicago sativa L.) breeding strategy to decrease slowly digestible or indigestible fiber and simultaneously increase digestible fiber could improve forage quality without reducing total fiber. The objectives were: (1) to estimate selection responses from divergent and opposite direction selections of (i) hemicellulose (HEM) and acid detergent fiber (ADF), (ii) acid detergent lignin (LIG) and HEM + cellulose (CEL) and (iii) CEL and HEM + LIG, and (2) to determine correlated responses in in vitro true digestibility (IVTD). Selection progress was evaluated in replicated plot trials at two locations, sampled for 2 or 3 yr. Selection for divergent HEM and ADF resulted in change only for ADF [10.9 g kg-1 dry matter (DM)]. Selection for divergent LIG and HEM + CEL, resulted in same direction change in LIG (3.3 g kg-1 DM). Selection for divergent CEL and HEM + LIG resulted in change only in CEL (5.1 g kg-1 DM). Low LIG and high HEM + CEL, and low ADF and high HEM populations had 9.7 and 8.3 g kg-1 DM higher IVTD than their counterparts, respectively. The first cycle of selection for the fiber components simultaneously in the opposite directions was not successful. However, reduced LIG or ADF concentration appears to increase alfalfa forage digestibility and decrease total fiber concentration. Key words: Alfalfa, cell wall, hemicellulose, cellulose, lignin, digestibility

A novel family of carbohydrate-binding modules identified with Ruminococcus albus proteins

We recently showed that some of the enzymes underpinning cellulose solubilization by Ruminococcus albus 8 lack the conventional type of dockerin module characteristic of cellulosomal proteins and instead, bear an “X” domain of unknown function at their C-termini. We have now subcloned and expressed six X domains and showed that five of them bind to xylan, chitin, microcrystalline and phosphoric-acid swollen cellulose, as well as more heterogenous substrates such as alfalfa cell walls, banana stem and wheat straw. The X domain that did not bind to these substrates was derived from a family-5 glycoside hydrolase (Cel5G), which possesses two X domains in tandem. Whereas the internal X domain failed to bind to the substrates, the recombinant dyad exhibited markedly enhanced binding relative to that observed for the C-terminal X domain alone. The evidence supports a distinctive carbohydrate-binding role of broad specificity for this type of domain, and we propose a novel family (designated family 37) of carbohydrate-binding modules that appear to be peculiar to R. albus.

Effect of industrial processing on alfalfa cell walls

Thermal treatment is one of the widely used methods for feedstuff preservation. The aim of this study was to evaluate possible cell wall alterations during the industrial-scale drying of alfalfa. Alfalfa is mainly dehydrated and pelleted using two drying treatments. First a thermal treatment at 70 °C is applied to the chopped raw material, prior to the main dehydration procedure which involves a temperature of about 600 °C for a restricted time (15 min). The different steps of the dehydration process did not cause significant changes in alfalfa cell wall composition, but the proportion of labile ether-linked lignin structure (β-O-4) increased after the thermal shock. Chemical fractionation of cell wall carbohydrates showed that pectins were less accessible to extraction with ammonium oxalate after alfalfa drying. Moreover, xylanase (endo enzyme) was more effective on cell walls obtained from heated material. These results suggested that heat processing causes some changes in interpolymer bonds (covalent and hydrogen bonds) between pectins and the lignin/hemicellulose matrix, whereby the cell wall network is modified. © 2002 Society of Chemical Industry

In vitro utilization of mucin, lung polymers, plant cell walls and insect cuticle by Aspergillus fumigatus, Metarhizium anisopliae and Haematonectria haematococca

Aspergillus fumigatus is saprotrophic with an unusual ability to colonize the respiratory tract. The mechanisms that permit pathogenicity may have evolved to adapt the fungus to life as a saprobe. To define the nature of these adaptations and identify common themes in fungal pathogenesis to vertebrates, insects and plants, we compared A. fumigatus with a plant pathogen (Haematonectria haematococca) and an insect pathogen (Metarhizium anisopliae) in their abilities to degrade and utilize host-derived macromolecules (horse lung polymers, porcine mucin, hyaluronic acid, alfalfa cell walls and cockroach cuticle). Each fungus produced a similar range of proteases on mucin and lung polymers, and high levels of several glycosidic enzymes on mucin and plant cell walls, which contain inductive carbohydrate substrates. Following 18 h of growth by A. fumigatus at pH 4 or pH 8, the degradation of mucin carbohydrates and mucin protein were approximately 40% and 75% respectively, suggesting that the aspartyl proteases (produced at pH 4) and the subtilisin proteases (produced at pH 8) are more important than carbohydrases for degrading mucin. The highly glycosylated mucin residue remaining after 18 h growth resisted further degradation, in part due to bound sialic acid as A. fumigatus secretes a sulphatase but not sialidase. Hyaluronidase activity (an important virulence factor in bacteria) was not produced by A. fumigatus, M. anisopliae or H. haematococca, but each fungus secreted a range of other enzymes (phospholipase A2, phospholipase C, acid phosphatase, alkali phosphatase, phosphodiesterase and esterase) that are common toxic components of bacteria as well as reptilia and invertebrate venoms. Thus thermotolerant opportunists such as A. fumigatus may sustain themselves and cause disease in human hosts using depolymerases that are widely distributed in fungi and that provide them with the versatility to exploit many environments.

Alfalfa Stem Tissues: Cell-wall Development and Lignification

Abstract Alfalfa stems contain a variety of tissues with different patterns of cell-wall development. Development of alfalfa cell walls was investigated after histochemical staining and with polarized light using light microscopy and scanning electron microscopy. Samples of the seventh internode, from the base of stems grown on cut stems, were harvested at five defined stages of development from early internode elongation through to late maturity. Internode seven was elongating up to the third sample harvest and internode diameter increased throughout the entire sampling period. Chlorenchyma, cambium, secondary phloem, primary xylem parenchyma and pith parenchyma stem tissues all had thin primary cell walls. Pith parenchyma underwent a small amount of cell-wall thickening and lignification during maturation. Collenchyma and primary phloem tissues developed partially thickened primary walls. In contrast to a recent report, the formation of a ring shaped, lignified portion of the primary wall in a number of cells in the exterior part of the primary phloem was found to precede the deposition of a thick, non-lignified secondary wall which was degradable by rumen microbes. In numerous xylem fibres from the fourth harvest date onwards, an additional highly degradable secondary wall layer was deposited against a previously deposited lignified and undegradable secondary wall. The pattern of lignification observed in alfalfa stem tissues suggests that polymerization of monolignols by peroxidases at the luminal border of the primary cell wall creates an impermeable zone which restricts lignification of the middle lamella region of tissues with thick primary walls.

An adhesion-defective mutant of Ruminococcus albus SY3 is impaired in its capability to degrade cellulose

An adhesion-defective mutant of Ruminococcus albus SY3 was isolated by a subtractive enrichment procedure, which involved repetitive adsorption of cellobiose-grown cells to cellulose. The growth characteristics of the mutant were compared with those of the wild type. Like the wild-type cells, the mutant was capable of growing on soluble substrates, i.e. cellobiose and xylan. However, in contrast to the wild type strain, the mutant was impaired in its capacity to utilize insoluble substrates, e.g. crystalline cellulose, acid-swollen cellulose or alfalfa cell walls. Scanning electron microscopy revealed protuberance-like surface structures on the wild-type strain which were absent on the mutant. The levels of endoglucanase and xylanase enzymatic activities released into the extracellular culture fluid were higher in the wild type compared to the mutant. However, Avicelase activity was not detected in the extracellular culture fluid of either strains when grown on cellobiose.

Nutritional ecology of dimorphic herbivores: digestion and passage rates in Nubian ibex.

We compared forage digestion and passage rates among three groups of Nubian ibex (Capra ibex nubiana) - mature males, non-lactating females, and lactating females - to test hypotheses relating intraspecific digestive ability to body mass and reproduction costs. We hypothesized that large males (60 kg) would exhibit longer forage retention times and more complete digestion of fermentable cell walls than adult females (23 kg). We tested these predictions by measuring digestion and retention of a grass hay and an alfalfa hay, forages that exhibited contrasting rates and extents of cell wall digestion. Consistent with predictions, males retained both forages longer than non-lactating females. However, by substantially increasing gut fill, lactating females increased both intake and retention time with respect to non-lactating females. Contrary to predictions, all three groups digested the grass (66% digestible) and alfalfa hay (63%) equally well. Alfalfa cell wall was less digestible than that of grass hay (60% vs 69% digestible), and retention time of alfalfa was consistently, but not statistically significantly, shorter. Fiber digestion was not correlated with retention time, emphasizing the ability of behavioral processes to modify digestion rate. We postulate that females achieved their greater digestion rate by masticating forages much more thoroughly than males.

Observations on In Situ Degradation of Forage Cell Components in Alfalfa and Italian Ryegrass

The rate and extent of degradation of forage feed fractions contained in alfalfa and Italian ryegrass hays were determined. Nylon bags filled with 4g of each forage were suspended in the rumen of two cannulated cows immediately before feeding and incubated for 10 different times (0, 2, 4, 8, 12, 24, 48, 72, 120, and 168h). The alfalfa hay, which had lower NDF, showed a lower extent, but a higher rate, of NDF degradation than the Italian ryegrass (41.1 vs. 59.8% and 4.64 vs. 2.91%/h, respectively). Alfalfa cell walls were degraded more rapidly than Italian ryegrass even though their lignin content was higher. The hemicellulose fermentation of alfalfa showed a longer lag time (13h) and an undegradable fraction nearly twice that for Italian ryegrass (63.3 vs. 37.1%). Cellulose from alfalfa was degraded at a higher rate than NDF or ADF, indicating that cellulose may be the primary site of hydrolysis of the cell wall in the rumen. Calculations based on in situ degradability indicate that alfalfa can have a higher inclusion than Italian ryegrass in diets for dairy cows because of lower NDF and greater availability of cell contents.

Digestion of cell-wall monosaccharides of ryegrass and alfalfa hays by the ruminal bacteria Fibrobacter succinogenes and Butyrivibrio fibrisolvens.

The ruminal bacteria Fibrobacter succinogenes strains S85 and BL2 were grown in monocultures or in coculture with strain D1 of Butyrivibrio fibrisolvens, and the solubilization of ryegrass and alfalfa cell walls (CW) and digestion of CW monosaccharides were measured. Fibrobacter succinogenes monocultures and cocultures with B. fibrisolvens D1 degraded 58-69% of ryegrass CW, solubilizing 67-78% of CW glucose, 65-71% of CW xylose, 69-75% of hemicellulose, and 68-77% of total CW monosaccharides. When grown on alfalfa CW, those cultures degraded 28-39% of alfalfa CW, solubilizing 42-58% of CW glucose, 30-36% of CW xylose, and 37-45% of hemicellulose. With respect to both substrates, F. succinogenes strains solubilized CW carbohydrates better than did B. fibrisolvens D1. Complementary interaction between B. fibrisolvens D1 and the F. succinogenes strains was identified with respect to the utilization of some solubilized carbohydrates, but not with respect to the extent of CW solubilization, which was determined mainly by the F. succinogenes strains. For both substrates, utilization of cellulose by F. succinogenes monocultures was high (96-98%), whereas that of hemicellulose was lower (24-26% in ryegrass and 49-50% in alfalfa). Under scanning electron microscopy, F. succinogenes bacterial cells attached to and colonized on CW particles were characterized by the appearance of protuberant surface structures that we have identified as "polycellulosome complexes."

Carbohydrate composition of alfalfa cell walls isolated from stem sections differing in maturity

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTCarbohydrate composition of alfalfa cell walls isolated from stem sections differing in maturityR. D. HatfieldCite this: J. Agric. Food Chem. 1992, 40, 3, 424–430Publication Date (Print):March 1, 1992Publication History Published online1 May 2002Published inissue 1 March 1992https://doi.org/10.1021/jf00015a012RIGHTS & PERMISSIONSArticle Views292Altmetric-Citations45LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (4 MB) Get e-Alerts Get e-Alerts

Digestion of Cell Wall Components by Dairy Heifers Fed Diets Based on Alfalfa and Chemically Treated Oat Hulls

Four Holstein heifers were used in a 4 × 4 Latin square design to measure total tract digestion of cell wall components from diets based on alfalfa haylage and alkaline hydrogen peroxide-treated oat hulls. Diets contained 90% forage and 10% concentrate. Treatments were diets containing 90, 70, 50, or 30% alfalfa haylage with treated oat hulls supplying the remainder of the forage portion. Total tract digestion of cell wall-associated uronic acids, arabinose, galactose, mannose, rhamnose, and lignin were not affected by forage source. Digestibilities of cell wall glucose and xylose increased with increasing level of dietary treated oat hulls, reflecting the positive effect of alkaline hydrogen peroxide treatment on cell wall digestion. Cellulose (ADF minus acid detergent lignin) digestibilities were similar to those for cell wall glucose, whereas hemicellulose (NDF minus ADF) digestibilities were similar to those for cell wall arabinose plus xylose. Low digestibilities of alfalfa cell wall xylose indicate that some cell wall structure inhibits the degradation of alfalfa xylans. Low degradabilities of core lignin, esterified p-coumaric acid, and esterified acetyl groups suggest that these components may be involved primarily in depressing fermentation of cell wall polysaccharides.

Effect of Forage to Concentrate Ratio and Intake Level on Utilization of Early Vegetative Alfalfa Silage by Dairy Cows

Two experiments were conducted to measure the effects of intake and forage: grain ratio on utilization of early maturity alfalfa silage in dairy cows. In Experiment 1, diets with three forage:concentrate ratios (percentage of silage, percentage NDF): low (56, 28.3), medium (71, 31.0), or high (86, 33.4) were fed ad libitum to six lactating, ruminally cannulated cows in a replicated 3×3 Latin square. The same diets were then fed at 1.3 × maintenance intake to six gestating dry cows. Daily milk yield and percentage and yield of milk protein and casein were higher for cows fed the low silage diet than for cows receiving other treatments. Fat percentage and yield were not different among diets. Lactating cows consumed more DM on low silage (23.0kg/d) than on medium or high silage diets (21.4kg), but NDF intake as percentage of BW was higher for the high silage diet. Digestibility of DM in the lactating (70.7, 69.9, and 67.5% for low, medium, and high) and dry cows (76.7, 73.5, and 69.0%, respectively) decreased as the level of silage increased. Depression in digestibility was greater as dietary concentrate increased. Cows fed the high silage diet had a faster fractional passage rate of solids and higher rumen fill. Digestion of concentrate cell walls appeared to be depressed more than alfalfa cell walls as intake increased.