In nutrition, the term fiber refers to the components of plant-derived foods and feedstuffs that are not digestible by mammalian enzyme systems (Moore and Hatfield 1994). In forages commonly fed to livestock, fiber refers to the plant cell wall. Mammals do not possess the enzymes to hydrolyze the predominant 1-4 linked polysaccharides that occur in cell walls and depend on microorganisms in the gastrointestinal tract to ferment these polysaccharides to absorbable nutrients. Ruminants are among the most specialized herbivores for utilizing this symbiotic relationship to exploit plant cell walls as a source of nutrients (Van Soest 1994). Obviously plant cell walls did not evolve to serve as a feed for ruminant animals. The biological functions of the cell wall have resulted in a structure that is of variable and often low digestibility by ruminants. Also, the physical volume occupied by cell walls in the rumen affects feed intake and animal performance. My objective in this review is to provide an overview and assessment of the methodologies available for the measurement of concentration, composition and structure of fiber or plant cell walls.

PHYSICAL AND CHEMICAL STRUCTURE OF PLANT CELL WALLS

During early development a plant cell must be able to grow in size (Iiyama et al. 1993). At this stage the cell wall is referred to as a primary wall and is capable of elongating because the wall polymers are not cross-linked. The middle lamella is the region between adjoining cells and is composed primarily of pectic substances, which are a group of galacturonan polymers with neutral sugars substitutions. Legumes contain large amounts of pectic substances, whereas grasses have relatively low concentrations. The primary wall consists of several polysaccharides, including cellulose, mixed linkage -glucans, heteroglucans, glucuronarabinoxylans and heteroxylans (Moore and Hatfield 1994). The xylans are much more abundant in grass walls than in legumes. Pectic polysaccharides of legumes are substituted with small amounts of ferulate and p-coumarate esters. In contrast, primary wall in grasses has high concentrations of ferulate esterified to arabinoxylans and lesser concentrations of p-coumarate esters. Structural and other proteins are also deposited in the primary wall of plants.

When the plant cell stops growing and initiates the maturation process, secondary wall deposition and lignification begin. Cellulose is the major polysaccharide deposited in the secondary wall. Grasses continue to deposit xylans; however, the degree of substitution of the xylans declines compared with these polysaccharides in the primary wall. 4-O-Methyl-glucuronoxylans are the major noncellulosic polysaccharides in secondary legume walls. Lignin deposition begins in the middle lamella and the primary wall (Terashima et al. 1993). Lignification then proceeds through the primary wall region and into the secondary wall, but lignin deposition always lags behind secondary wall polysaccharide deposition. The result of this pattern of development is that the most recently deposited polysaccharide in the wall is not lignified. Lignin is a polymer composed of cinnamyl alcohol precursors. Guaiacyl lignin derived from coniferyl alcohol monolignols is the predominant type of lignin in the primary wall, whereas the secondary wall is relatively richer in syringyl lignin formed from sinapyl alcohol precursors.

Lignin is covalently bound to cell wall polysaccharides, but until recently little was known about the form of this cross-linkage. In grasses, ferulate esters of arabinoxylans react with monolignols to form cross-links between polysaccharides and lignin (Ralph et al. 1995). Ferulate esters become incorporated into the lignin polymer through ether and C-C bonds. Data suggest that these ferulate units may serve as the initiation site for lignification in grasses. The cross-linkage structure in legumes has not been identified and is unlikely to be ferulate esters, given their low concentration in legume cell walls. However, tyrosine residues in wall proteins could serve a similar cross-linking role, and legumes have large amounts of primary wall protein (Bacic et al. 1988). Grasses also deposit large amounts of p-coumarate as -esters of syringyl lignin during later stages of lignification (Ralph et al. 1994).

The developmental profile outlined above is for a vascular tissue cell, such as sclerenchyma, which goes through extensive secondary wall thickening and lignification (Wilson 1993). Mesophyll and parenchyma cells accumulate very little secondary wall or lignin. Cell wall concentration, composition and digestibility of plant tissue types differ dramatically as a result. Although it is possible to analyze the chemical composition of individual plant tissues, the required isolation procedures preclude routine use of such methods. As a result, fiber analysis can provide only an average composition of the cell walls in a feed and cannot account for the influence of individual tissue types on ruminant performance.

METHODS OF FIBER ANALYSIS

Table 1. Uses and limitations for some of the major methods of fiber and cell wall analysis used in forage quality and ruminant nutrition [View Table]

In ruminant nutrition the neutral detergent fiber (NDF) method developed by Van Soest has largely replaced CF (Van Soest 1963b). Neutral detergent fiber, like CF, uses chemical extraction (with a neutral detergent solution under reflux) followed by gravimetric determination of the fiber residue. Neutral detergent fiber is considered to be the entire fiber fraction of the feed, but it is known to underestimate cell wall concentration because most of the pectic substances in the wall are solubilized (Van Soest 1994). As a result, NDF is a poor estimate of cell wall concentration for the pectin-rich legumes. Heat-damaged proteins in processed feeds are also retained in NDF, which will overestimate fiber content. These shortcomings of NDF as a method to determine cell wall concentration are certainly a problem if one is interested in the plant cell wall as a biological structure, but as pointed out by Van Soest (1994) these inconsistencies are not of concern if fiber is defined as the incompletely digestible fraction of feeds. Although widely used for fiber analysis of ruminant feeds, the NDF procedure is not an official AOAC method. Acid detergent fiber (ADF) is a portion of the plant fiber (Van Soest 1963a). Acid detergent fiber includes the cellulose and lignin from cell walls and variable amounts of xylans and other components. Acid detergent fiber is an AOAC-approved method of analysis. A common variation of the ADF method is to use NDF as a pretreatment (Van Soest and Robertson 1980).

The Prosky dietary fiber (DF) procedure is the third major gravimetric method for measuring fiber concentration (Prosky et al. 1984). This method utilizes a series of enzymatic and chemical pretreatments followed by precipitation in 80% ethanol to isolate a fiber residue. Unlike NDF and CF, the DF method retains all the cell wall components. Starch and protein removal can be a problem in concentrate and processed feeds. Incomplete removal of these substances will result in DF overestimating cell wall concentration. The Prosky method has been recognized as an official AOAC method.

Many analytical methods exist for the measurement of specific cell wall components. In ruminant nutrition, forage cellulose and hemicellulose concentrations are commonly estimated as ADF minus sulfuric acid detergent lignin (ADL) and as NDF minus ADF, respectively (Van Soest and Robertson 1980). Cellulose concentrations are overestimated by ADF minus ADL to the extent that xylans are present in ADF and underestimated by heat-damaged protein contamination of ADL. Similarly, hemicellulose estimates based on NDF minus ADF are overestimated by non-extracted protein in NDF, and residual xylans in ADF cause underestimates of hemicellulose. The Crampton-Maynard method is thought to provide a better measurement of cellulose in forages (Morrison 1980), and trifluoroacetic acid hydrolysis of NDF can be used to determine the hemicellulosic sugars (Moore and Hatfield 1994). Pectin content of forages can be calculated from the summation of uronic acids and the major pectic neutral sugars, but this approach suffers from the inability to separately measure galacturonic and glucuronic acid residues. Alternatively, pectin can be extracted and determined gravimetrically or after hydrolysis to its sugar components. Very sophisticated extraction schemes have been developed to isolate specific classes of polysaccharides (Moore and Hatfield 1994). These isolated polysaccharides can be characterized for sugar composition, linkage patterns by methylation, and specific molecular structures using NMR. Although more data on the specific polysaccharide structures in forage cell walls are becoming available, these data are not directly utilized in ruminant nutrition.

Many methods of lignin analysis have been developed because of lignin's negative association with digestibility. Acid detergent lignin is the most common lignin method in ruminant nutrition, but increasing evidence indicates it underestimates lignin due to solubilization of some lignin at the ADF step in the procedure (Lowry et al. 1994). Klason lignin is the oldest lignin method and has been considered inappropriate for forages because of protein contamination. Recent results indicate this concern is unnecessary, and Klason lignin may be the best method available for estimating lignin content (Hatfield et al. 1994). The previous two lignin methods measure the residue remaining after acid hydrolysis. The permanganate, sodium chlorite and acetyl bromide lignin methods solublize the lignin and measure lignin as either weight loss or by spectrometry (Collings et al. 1978, Morrison 1972, Van Soest and Wine 1968). These oxidative methods are susceptible to incomplete solubilization of lignin or interference by other solubilized wall components. Lignin composition can be determined by nitrobenzene oxidation, thioacidolysis and pyrolysis of forages (Lapierre 1993). Chromatography is required for identification of the lignin degradation products from these methods, and they all suffer from lack of information on completeness of recovery of the lignin components. The ester-linked ferulic and p-coumaric acids in the cell wall can be extracted by alkaline hydrolysis at low temperature (Hartley 1972). Those ferulate molecules involved in ether cross-links between wall polysaccharides and lignin are hydrolyzed by high temperature alkaline treatment (Iiyama et al. 1990). Dioxane-HCl treatment is an alternative method for cleaving etherified hydroxycinnamic acids (Scalbert et al. 1985). Chromatography is used to identify and quantify these hydroxycinnamic acids.

FUTURE TRENDS IN FIBER ANALYSIS

In the research setting, most scientists are more interested in comparing treatments than generating absolute values. The opposite situation exists in the feed analysis industry. The plethora of methods and variations on specific methods developed in the research environment cannot be transferred to industry. Standardization of methods through organizations such as AOAC and the National Forage Testing Association will be needed even more in the future as producers utilize forage testing more extensively as an aid in diet formulation. Establishment of an "official" method of NDF analysis is probably the most critical need because of the central role NDF values have assumed in ruminant diet formulation. Similarly, although I have not discussed methods of measuring fiber digestibility, it will be necessary to standardize an in vitro or in situ method for this forage trait.

Sophisticated measurements of cell wall composition and structure will become more prevalent in research settings where information on cell wall development and composition and the mechanisms of ruminal degradation of cell walls is the objective. Use of these analytical methods in research more closely related to applied animal nutrition will be limited. Before they are widely adopted, these methods must demonstrate that they provide information that can improve the accuracy with which we predict animal performance. Examples of possible improvements would be if inclusion of pectin in the fiber fraction increases predictive power for intake or if polysaccharide composition information improves understanding of rates and extent of fiber digestion. These more sophisticated analytical methods may see their first practical use in the plant improvement field, in which genetic changes are targeted. Specific molecular components should be more closely tied to genes than empirical traits such as NDF.