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Jess D Reed
International Livestock Centre For Africa
Addis Ababa, Ethiopia
Peter J Van Soest
Department of Animal Science
Cornell University
Ithaca, New York, U S A


The detergent system of forage analysis is based on the logic of the Lucas test for feed fractions with uniform nutritive availability. The detergent system is flexible in application and can supply important information about the nutritive value of crop residues and agro-industrial by-products. Heat damaged proteins, lignin, biogenic silica, total fibre and cutin can all be estimated by application of the detergent system.
Condensed tannins can form protein complexes in the fibre complexes. Methods are described for investigating this problem.
The detergent system can be used to study alkali-treated straw by measuring the optical density of neutral detergent solubles and sequential extraction by neutral-detergent followed by acid-detergent to determine lignin.


The detergent system of analysis was designed to replace the Weende system (proximate analysis) for estimating the nutritive value of fibrous feeds by chemical methods. The failure of proximate analysis to separate feeds into meaningful nutritive fractions has been reviewed elsewhere (Van Soest and Robertson 1980; Van Soest 1967). This paper will discuss the biological and nutritional meaning of the detergent system and problems associated with interfering plant compounds in relationship to the analysis of crop residues and agro-industrial by-products.

The most fundamental concept of the detergent system is based on the separation of feeds into fractions with uniform or non-uniform nutritive availability as defined by the Lucas test (Lucas et al 1961; Van Soest 1967). The test for uniform nutritive availability is based on analysis of digestible amount of a feed fraction regressed on the percent of the fraction in the feed. For feed fractions that are represented in the faeces by indigestible feed, microbial debris and endogenous excretion, the slope represents true digestibility and the negative intercept estimates the metabolic amount as a percent of intake. A feed fraction with uniform nutritive availability has a regression equation with a low standard error and an intercept less than or equal to zero. A high correlation coefficient is not indicative of uniform nutritive availability.

Cell contents and protein are feed fractions which usually have uniform nutritive availability. Both feed fractions have true digestibility greater than 90% and negative intercepts (Figure 1). Lignin is a feed fraction with uniform nutritive availability, but has a true digestibility, intercept and correlation coefficient not significantly different than zero (Figure 2).

Cell wall, cellulose and hemicellulose are feed fractions with non-uniform nutritive availability (Figures 3 and 4). They have regression equations with high standard errors (Table 1). Negative intercepts have no biological meaning because there can be no endogenous excretion of cell wall carbohydrates.

Figure 1

Figure 1:
Lucas test for the uniform nutritive availability of neutral-detergent solubles (NDS) (cell contents). The regression equation has a slope of 0.98 indicating true digestibility of cell contents is almost complete. The negative intercept is 12.9 which is an estimate of the endogenous metabolic excretion as a percent of intake.

Figure 2

Figure 2:
The Lucas test for 72% H2 SO4 lignin indicating uniform nutritive availability. The intercept, slope and correlation are not significantly different from zero but the regression equation has a low standard error

The apparent digestible amount of uniform feed fractions can be predicted by subtracting an estimate of metabolic amount from the product of true digestibility and amount in the feed. This approach can be misleading when applied to by-products in which heating or fermentation has caused heat damaged protein, in crop residues that contain inhibitory compounds such as tannins and in feeds treated with alkali to increase digestibility (Parra, this symposium). An important aspect of the detergent system is the ability to predict deviations from uniform nutritive availability in cases where heat damage or inhibitory compounds are suspected.

Figure 3

Figure 3:
Lucas test for hemicellulose showing non-uniform nutritive availability. Individual regressions for grasses and legumes have positive intercepts which have no biological meaning. The overall regression has a high correlation (0.94) caused by the interaction between hemicellulose contents in grasses versus legumes and does not indicate uniformity.

Table 1 Summary of the application of the Lucas test to various feed fractions for estimating true digestibility. Endogenous excretions different from zero for fibre and fibre fractions have no biological meaning because there is no digestive tract source of fibre (from Van Soest 1967; Arroya and Evans 1972; Parra 1978; Aerts et al 1978)
FractionMean apparent dig (%)True digestibility (%)Endogenous excretion (%)
Cellular contents
Cell wall nitrogen86.07
Cell wall (NDF)   
Range43–5952–68-11 to +11
Range47–6636–79-5 to +2
Range45–6343–73-5 to +4
Lignin (Klason)
Range1–2-2 to +3-.1 to +.5
Lignin (permanganate)20482
Crude fibre47–5536–61-7 to +2
NFE55–7255–61-4 to +1
Figure 4

Figure 4:
Lucas test for cellulose showing non-uniform nutritive availability. The slope is 50% with a large standard error (± 13.5)


The separation between uniform feed fractions and non-uniform feed fractions is achieved by neutral detergent extraction which separates cell contents (uniform) from cell wall (non-uniform) in feeds and bacterial and endogenous debris from undigested cell wall in faeces (Mason 1979). Neutral-detergent fibre (NDF) consists of cellulose, hemicellulose and lignin, which represent the major cell wall fractions. Biogenic silica and pectins are not recovered in NDF, but soil silica, heat damaged proteins and tannin protein complexes are recovered (Figure 5).

Starch in cereal grains and milling by-products interferes with neutral-detergent extraction by forming stable gels causing filtering problems. The NDF procedure has been modified by using heat stable amylase to avoid this problem, reducing the error of determination and the amount of NDF (Robertson and Van Soest 1977; Schaller 1976).

Although NDF recovers the true indigestible feed fraction in faeces it is not highly correlated with digestibility (Table 2). NDF digestibility depends

Table 2 Correlation between digestibility, intake and fibre fractions. Acid detergent fibre and lignin have the highest negative correlation with digestibility while neutral detergent fibre (cell wall) has the best correlation with intake
Digestibility (in vivo) --+.61
Digestibility (in vitro)a+.80+.47
Acid detergent fibre-.75-.61
Crude protein+.44+.56
Cell wall-.45-.76

a Two stage procedure of Tilley-Terry asmodified by Goering and Van Soest (1970)

Figure 5

Figure 5:
Flow chart of analytical sequences of detergent analysis illustrating its flexibility for several important feed fractions. The system has evolved to include many fractions not considered in the original scheme. Depending on the material to be analysed and the objectives of the analysis, the detergent system can be used in many different ways.

On lignin and silica content, rate of passage and rate of digestion, and physical and chemical properties of the cell wall carbohydrates. In many crop residues, especially from cereals, NDF is the most important substrate for rumen fermentation. However NDF has a strong negative correlation with intake (Table 2). The correlation can be improved by correcting for differences between individual intake relative to a common forage (Figure 7) (Osbourn et al 1974). The physical characteristics of fibre may limit intake by rumen fill (Van Soest 1982) and pushing rumination time to its limit (Welch and Smith 1969).


Acid-detergent fibre (ADF) is a preparatory extraction for the determination of cellulose, lignin, silica and heat damaged protein (Van Soest and Wine, 1967). Pectin is recovered in the ADF. In pectin containing crop residues such as sweet potato vines the amount of ADF equals NDF (Table 4). This interference can be avoided if acid-detergent extraction is conducted sequentially after neutral-detergent extraction.

An estimate of heat damaged protein is made by a determination of nitrogen in the ADF by Kjeldahl (Figure 5). The Maillard reaction is the polymerization of protein with carbohydrates (mainly pentoses in hemicellulose) created by heating due to industrial processes or fermentation. These heat damaged proteins are indigestible. Oil seed meals usually have low ADF nitrogen when oils are extracted by press or solvent without heating. Other by-products can have high ADF nitrogen such as brewary dried grains, tomato pomace and by-products from the extraction of essential oils (Wohet et al 1981) (Table 3). Oven drying for feed analysis at temperatures above 60°C can result in heat damaged protein and elevated fibre and lignin values.

Figure 6

Figure 6:
Relationship between bepsin-insoluble neutral-detergent fibre - crude protein (NDF-CP) and neutral-detergent insoluble condensed tannins.

TABLE 3 Acid-detergent fibre bound nitrogen in some agro-industrial by-products
 Total Nitrogen (% DM)ADF bound nitrogen (% of total nitrogen)
Noog meal4.543.9
Tomato pomace3.7038.6
Distilled dried grain4.2718.8
Nutmeg hulls*1.4087.2
Cinnamon bark*0.64100.0
Black Pepper*2.6451.4
Vanilla bean*1.1464.0
Ginger root*1.2836.7
Coffee bean*2.5839.5
Cocoa bean*4.3239.7
Celery seed*3.1822.2

* Data compiled from Wohet et al (1981)

Lignin is a phenolic polymer that lowers digestibility of cell wall carbohydrates. Early lignin methods used 72% H2SO4 treatment of complete forage for lignin determination. Protein interferes with these Klason lignins. Acid detergent extraction removes all protein except those that are closely associated with lignin or heat damaged. Treatment of ADF with 72% H2SO4 removes cellulose (Figure 6). The organic residue is mostly lignin unless the feed contains heat damaged protein or cutin. Oxidation of lignin with KMnO4 leaves an organic residue of cellulose and cutin. Sequential treatment of ADF with KMnO4 and 72% H2SO4 leaves an organic residue of cutin.

Silica is quantitatively recovered in the ADF ash which can be treated with HBr to remove other acid insoluble minerals. Biogenic silica lowers digestibility between 1.4 and 3.0 units for each unit of silica. Soil silica is also recovered in the ADF ash but has no effect on digestibility except as an inert diluent.

Attempts to use ADF as a replacement for crude fibre are totally erroneous and suffer from the same problems because ADF does not recover hemicellulose (Van Soest and Robertson 1980). Since ADF is a preparatory step, its use in predicting digestibility is not consistent with the logic of detergent analysis.


Condensed tannins can form protein complexes that are insoluble in neutral-detergent. Condensed tannins are also termed proanthocyanindins because they yield anthocyanidins, which are red pigments that form in hot solutions (Bate-Smith 1973). The solubility of proanthocyanidins is a function of chemical structure. Prodelphinidins are the least soluble and form strong protein complexes. Methods of sample preparation and extraction will also influence amount of neutral-detergent insoluble proanthocyanidins. Oven drying feeds that contain proanthocyanidins, even at temperatures below 60°C, increases NDF, fibre bound nitrogen and lignin (Reed et al 1984). proanthocyanidins in NDF can be detected by heating NDF in 5% HCl in n-butanol yielding a red solution from which absorbance can be read at 550 nm for a semi-quantitative estimate of amount. Cassava leaves contain proanthocyanidins in the NDF that are highly correlated with the amount of pepsin insoluble NDF nitrogen (Figure 6)(Reed et al 1982).


Treating straw with alkali saponifies phenolic esters between phenylpropanoids (lignin) and hemicellulosic carbohydrates (Hartley and Jones 1978). If the straw is rinsed, soluble hemicellulose and phenolics are removed. These soluble cell wall components are also removed by neutral-detergent. Since phenolics absorb UV light, the optical density at 280 nm of the neutral-detergent solution can be used to test the effectiveness of alkali treatment (Figure 8) (Lau and Van Soest 1981). The saponification of lignin from the NDF of treated straw can also be used to study the effectiveness of treatment (Figures 8 and 9).

Acid-detergent lignin does not decrease upon alkali treatment without rinsing because the saponified phenolics may not be soluble at low pH (Rexen and Vestegaard Thomsen 1976). However, a sequential extraction by neutral detergent followed by acid detergent will lower the lignin value in relation to increased effectiveness of alkali treatment and increased digestibility (Van Soest 1981). Pre-extraction of unrinsed alkali treated straw with water buffered at pH 7 followed by acid-detergent extraction should have the same effect on lignin values.


The summative equation is based on the logic of the Lucas test for uniform nutritive availability. Cell contents have a true digestibility of 98%. Digestibility of cell wall is estimated by the lignin to ADF ratio (Goering and Van Soest 1970). The sum of digestible cell wall and digestible cell contents is an estimate of true digestibility. Apparent digestibility is obtained by subtracting an estimate of the metabolic amount from true digestibility. A correction for the effects of silica on digestibility is applied when silica is greater than 2%. For 34 straw samples from the Ethiopian Highlands the summative equation gave the same estimate of apparent digestibility as Tilley and Terry in vitro digestibility (Table 4). A metabolic amount of 12.9 and a silica correction factor of 1.4 were used in the summative equation.

Fibre quality as affected by lignin and silica is an important aspect in the utilization of cereal crop residues. The NDF is usually greater than 70% of the dry matter with small species variation (Table 5). The lignin content of cereal crop residues is low compared with crop residues from dicotyledons, thus potentially digestible fibre in cereal crop residues is greater. Potential digestibility of fibre in cereal crop residues may not be reached because nitrogen and other microbial nutrients are limiting.

TABLE 4 Comparisons of Tilley and Terry in vitro digestibility and the summative equation in estimating apparent digestible dry matter (ADDM) of 34 Ethiopian straw samples
ADDM summative equationADDM Tilley and Terry in vitroPaired t-testdifference

* not significant at the 5% level of probability

TABLE 5 Nitrogen, neutral-detergent fibre (NDF), acid-detergent fibre (ADF), lignin (LS), silica and and apparent digestible dry matter (ADDM) in some important cereal crop residues. ADDM was estimated by the summative equation
Maize upper stalk0.4175.844.
Maize leaf sheath0.3383.947.
Maize tassel0.7982.346.
Sorghum leaf sheath0.2579.953.
Millet leaf sheath0.3274.947.84.11.964.2
Teff straw0.5173.544.24.23.360.0
Wheat straw0.4172.
Rice straw0.7771.
Rice hulls0.6680.080.815.622.911.3

The quality of dicotyledon crop residues can be highly variable (Table 6). The horse bean haulm sample in Table 6 has a high fibre and lignin content and a low nitrogen and digestibility, indicating a large proportion of stem. Leafy sweet potato vines and cassava tops have higher nutritive value because of lower NDF and higher nitrogen. In the case of cassava leaves part of the nitrogen is unavailable because of tannins. Mango leaves are an interesting example because they contain a high level of biogenic silica which is unusual for dicotyledons. Mango leaves also contain tannins. The combination of both tannin and silica may account for the low in vitro digestibility.

TABLE 6 Nitrogen, neutral-detergent fibre (NDF), acid-detergent fibre (ADF), lignin (LS), silica and apparent digestible dry matter in some non-cereal crop residues. ADDM was estimated by the summative equation for horse bean haulms and by Tilley and Terry in vitro digestibility for the other samples.
Horse bean haulms1.1173.363.814.4-43.0
Sweet potato vines1.5639.339.
Cassava leaves3.9739.625.98.90.566.1
Mango leaves1.0639.339.012.35.646.5
Figure 7

Figure 7:
Relationship between intake and neutral-detergent fibre (cell wall). Lower figure shows the effect of increasing the correlation by correcting the data for differences in appetite (of sheep) (Relative intake) (r= -0.83 for upper figure versus r= -0.88 for the lower figure) (Osbourn et al 1974)

Figure 8

Figure 8:
Relationship between ultraviolet absorbance of neutral detergent (ND) extracts (solid line) and of sodium hydroxide extract of neutral-detergent fibre of alkali treated straw with in vitro digestibility (IVRD). The more effective treatments have higher UV absorbance of ND extracts, but there is a point where further increases in saponification of phenolics no longer increases in vitro digestibility (Lau and Van Soest 1981).

Figure 9

Figure 9: Relationship between saponifiable groups in the neutral-detergent fibre and in vitro digestibility (IVRD) of alkali treated straw. The more effective treatments have fewer saponifiable groups because the original alkali treatments have removed phenolic esters leading to increased UV absorbance of ND extracts (see Figure 8) (Lau and Van Soest 1981).


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A Nefzaoui and M Vanbelle

Laboratoire de Biochimie de la Nutrition
Université Catholique de Louvain, Place Croix du Sud
3–1348 Louvain - la - Néuve, Belgium


The in vitro digestibility technique of Tilley and Terry is the best available at present for predicting the nutritive value of forges and by-products in spite of some obvious disadvantages:
Enzyme techniques, despite slightly lower precision, offer several advantages:
Its major disadvantage consists in the difficulty of obtaining enzyme brands of large spectrum of action with constant activity from one batch to another.
To obtain less variable results among several laboratories it would be necessary to:
Chemical and in vitro methods are complementary. The adaptation of the fractional separation technique of Van Soest to non-conventional crop residues and agro-industrial by-products constitutes a priority for discerning the problems relative to the understanding of the cell wall indigestible fraction.
The information about the environmental conditions (eg: temperature, rainfall) the species, the state of development, can be more reliable than a simple chemical criterion.
In vitro determinations destined to predict the digestibility of feeds are not absolute measurements, and are more appropriately used for screening purposes.


Climatic differences between temperate and subtropical or tropical regions act directly upon the nutritive value of forages and by-products (Goto and Minson 1977; Van Soest 1982). Among these differences, high temperatures have the clearest impact on the chemical composition and especially on the cell wall components. A rise in temperature involves enrichment in the plant cell wall components (Deinum et al 1968; Marten 1970; Wilson and Ford 1971; Faix 1974; Guessous 1981; Van Soest 1982). The latter act directly on the digestibility and on the intake of forages. On an average, a reduction in digestibility of 0.6, 0.56, 0.28 and 0.21 points is ascribed to an average daily increase in temperature of 1°C for the tropical and temperate grasses and legumes respectively (Guessous 1983). However, such a depressant action of the temperature on the digestibility should be lowered by the positive effect of light. Yet, this effect remains limited compared to that of the temperature (Wilson 1981). Both the content and the nature of the cell wall influence the rate of digestion and the retention time of particles in the rumen. These are two decisive factors influencing the level of intake (Mertens 1977). The quality or the feeding value of forages or of by-products is represented by the intake and the digestibility. Predictability of digestibility is above all a function of the indigestible fraction of the cell wall.

TABLE 1 Effects of type of incubation vessel, tube position and agitation on in vitro dry matter digestibility (means and SE) (from Mellenberger et al 1970
 Aspen woodCotton lintersAmmoniated aspen wood
Type of tube
* valved11 ± 6.540 ± 13.533 ± 10.8
* sealed20 ± 2.963 ± 5.437 ± 2.1
Tube position
* horizontal
• shaken23 ± 0.775 ± 2.5--
• not shaken24 ± 1.470 ± 7.4--
* vertical
• shaken19 ± 1.861 ± 5.1--
• not shaken15 ± 3.149 ± 14.0--
* rotated18 ± 4.545 ± 6.2

Figure 1

Figure 1:
Flow diagram of the Van Soest modification to Tilley-Terry in vitro digestibility

Tropical or subtropical regions are characterized by:

The determination of in vivo digestibility is long and costly and it necessitates indirect methods if one wishes to arrive quickly at concrete recommendations. Numerous literature reviews have been published as to the choice of the method to be applied (Johnson 1966; Morrison 1976; Van Soest 1976; Osbourn 1977; Barnes and Marten 1979; Jarrige 1980; Osbourn and Siddons 1980; Van Soest and Robertson 1980; Demarquilly and Jarrige 1981).


“In vitro” fermentation techniques with rumen juice

“In vitro” investigation methods were developed long ago (Moxon and Bentley 1955; Bentley 1959; Johnson 1963). These methods differ in the preparation of rumen juice, in the respective proportions of the forage and the liquid phase, in the operational technique, in the incubation time and in the measurement of the digested constituents (DM, OM, cellulose). All have been supplanted by the two stage technique of Tilley and Terry (1963), which appears to be the most useful. In any case, the principle is always the same. The sample is put in a glass container in the presence of rumen juice (inoculum) and where the conditions are as similar as possible to those of the rumen. The amount of dry matter (DM), organic matter (OM) or cellulose which disappears after a certain incubation time is “in vitro” digestibility and this is related with “in vivo” data (digestibility and intake…). Thus this method is based on an insoluble residue.

Buffer solutions and nutritional media: Most of the utilized media are based on the “solution of McDougall” which corresponds to the artificial saliva reconstituted from the analyses of sheep saliva (McDougall 1949). But for some special reasons, certain modifications were introduced: addition of oligo- elements (Burroughs et al 1950), increase in buffer strength to avoid readjustment of the pH (Donefer et al 1960) and addition of valeric acid and biotin in the digestion studies of cellulose (Bentley et al 1954; Bryant and Doetsch 1955).

Different proportions of inoculum/buffer are used by various researchers and the ratio 1:1 seems to be the most adequate for feeds of low digestibility. Thus, Mellenberger et al (1970) have recorded in vitro DMD of aspen wood as 21.2, 23.9, 4.6 and 2.0% when the ratios of inoculum: buffer are 5:1, 4:1, 1:1, 1:4 and 1:10 respectively.

Glassware, gaseous phase, agitation, tube position: Any glass container can be used if the anaerobic conditions are respected. It is recommended especially in the studies of cellulosic materials to use equipment which reduces to the strict minimum the transference of material from one container to the other. Thus the use of centrifugation tubes which serve for the incubation and for the analysis after centrifugation reduces considerably the sources of error. The anaerobic conditions necessary for normal activity of the rumen microorganisms are ensured by saturating the medium with Co2. This gasification can be conducted differently, either in sealed or valved systems. The system with continuous passing of CO2 involves costly and complicated equipment. Between the two systems, valved or sealed, the data seem to indicate (Table 1) that the values obtained with the sealed system are higher and more accurate.

The modification by Van Soest (Goering and Van Soest 1970) (Figure 1) uses a CO2 manifold to flush 125 ml Erlenmeyer flasks. The substrate, buffer and microbial nutrient media are placed in the flask and flushed with CO2. A reducing agent and redox indicator ensure complete anaerobic conditions. Rumen contents (approximately 50% solid digesta and 50% fluid) are blended under CO2 to remove fibre-digesting organisms from the fibre, filtered under CO2, cheesecloth and glass wool. Ten ml of the inoculum are delivered into the flasks that have become anaerobic and the flasks are sealed. A manometer allows detection of leaks and ensures positive pressure from gas produced in the fermentation; CO2 is not continuously flushed through the system.

It is generally accepted that the agitation of the tubes during incubation, even with insoluble substrates, is not necessary. However, the results of Mellenberger et al (Table 1) show that the horizontal position of the tubes with agitation ensures the most elevated digestibilities and the lowest standard errors. This is very important for poor feeds.

Temperature: Usually the temperature is maintained at 39°C and a water bath with constant temperature (± 0.5°C maximum) is recommended. Temperature variations as little as 0.5°C can make the comparisons among the series difficult. It should be remembered that the rumen bacteria are very sensitive to elevated temperatures which make them lose their activity; hence it is necessary to see that the increase in temperature does not exceed 40°C.

pH: The optimum pH for cellulose digestion has been found to be 6.9 (6.7–7.0). The pH changes often during the fermentation process and it is sometimes necessary to readjust it. This readjustment depends on the rate, the extent and the type of fermentation (Johnson 1966). During cellulose digestion the production of acids is less than during starch digestion.

Incubation time: This depends on the aim of the study. Different times may be used according to the objectives of the fermentation (eg: the maximum rate of digestion and the type of substrate. Soluble sugars are very rapidly fermented and reach their activity peaks after one to two hours, whereas cellulose digestion begins only after a long incubation time and with a lower rate of digestion. In the sealed systems, it is difficult to maintain the maximum rate of fermentation for a long period because the substrate becomes limiting. Also the possible feedback effects of the end-products must be considered. Several incubation times (6, 12, 24, 48 h and more) are used by different researchers. However, for feeds with low digestibility, it is necessary to extend the incubation time beyond 48 h because, in addition to the rise in digestibility value, the variation is diminished (Table 2).

TABLE 2 Effect of incubation time on extent and variation of in vitro dry matter digestibility (Mellenberger et al 1970)
 48 hours incubation Coefficient96 hours incubation Coefficient
Digestibility (%)of variationDigestibility (%)of variation
Within dayBetween daysWithin dayBetween days
Aspen wood18.312.917.930.35.04.7
Ammoniated aspen wood40.04.610.347.92.83.3
Cotton linters73.74.311.

The technique of 6 hr “in vitro” rumen fermentation has been utilized by Allinson et al (1969) for determining the nutritive value of species of medicago, and by Ingalls (1964) and Crampton et al (1960) who showed that this technique gave values that were positively correlated with the nutritive value index (relative intake × digestibility). Since then, several authors have utilized the 6 hr degradability for predicting the intake and the nutritive value.

The source of inoculum: All the authors agree to the fact that the influence of rumen juice on in vitro digestibility measurements is the most difficult factor to control. The rumen has the advantage of being abundantly supplied with bacterial and protozoan species, but an inconvenience also results from the fact that these species change very rapidly in their proportions. These changes are due, among other factors, to the ingested ration. Moreover, animal species can influence the rumen juice activity. Several authors have emphasised that there are few differences between the rumen juice activity of sheep and cattle, as long as the two species are given the same diet (Lefevre and Kamstra 1960; Van Dyne and Weir 1964; Smith et al 1965; Drew 1966; Engels et al 1970; Van der Koelken and Dijkstr a 1971). Grant et al (1974) have concluded that after a sufficiently long time of adaptation to the ration, there was no significant difference between the rumen liquid of buffalo and that of Philippino and American dairy cows. Scales et al (1974) have shown that the inoculum of bovines is less variable than that of sheep and that the prediction equations established with the rumen juice of bovines are more precise than when the inoculum comes from sheep. On the contrary, Van Dyne and Weir (1964) have mentioned a greater variability of rumen juice activity in bulls than in sheep.

The diurnal variations of the rumen liquid activity seem to be more important than those among animals (Troelsen and Hanel 1966; Bowden and Church 1962). Opinions differ also concerning the influence of the moment of the sampling of the rumen liquid on its activity. Den Braver and Eriksson (1967) did not notice any significant difference, whereas Baumgardt et al (1962) and Wilkins (1966) are of the opinion that the moment of sampling is important. Inoculum activity diminishes and its variability increases as the sampling is delayed after feeding (Tilley and Terry 1963). The optimum dynamic conditions of the inoculum were thought to be two to four hours after feeding (Cottyn and Boucque 1968).

The influence of the ration of the donor animal on the inoculum activity is also very controversial. Some think that this factor has little or no importance (Salsbury et al 1958; Barnett 1957; Stewart and Schultz 1958; Quicke et al 1959; Walker 1959; Tilley et al 1960; Den Braver and Eriksson 1967). Others, on the contrary, are of the opinion that the ration of the donor animal can exert an important influence on the inoculum activity (Baumgards et al 1962; Asplund et al 1958; Reid et al 1959, 1960; Hungate et al 1960; Van Dyne, 1962; Barnes 1965; Bezeau 1965; Johnson 1969; Calder 1970; Nelson et al, 1972; Minson and McLeod, 1972). Tilley and Terry (1963) and Raymond and Terry (1966) have observed that only rations with an extremely low protein content had an influence on rumen juice activity. Nelson et al (1972) noticed that the effect of the ration of the donor animal tends to disappear if glucose and urea are added to the inoculum. Knipfel and Troelsen (1966) reported that the effect fades away when the incubation time passes from 12 to 48 h. Several researchers suggest that the diet of the donor animal should be identical with that of the substrate to be tested (Bryant and Burkey 1953; Church and Peterson 1960; Warner 1965; Johnson 1966). In any case it is preferable to use the rumen juice of several animals in each series, than of one alone, and to standardise the type of ration over the whole year. It is also important to sample the rumen fluid at fixed hours of the day (2–4 h after the morning feeding).

Preparation of the samples: Sample size does not seem to be a very important factor (McLeod and Minson 1978). An increase in sample size is followed by a slight reduction in digestibility and an improvement of the standard deviation (Table 3).

TABLE 3 Effect of pre-wetting and of sample size on the in vitro dry matter digestibility (means and SE) (from Mellenberger et al 1970)
 Aspen woodCotton lintersAlfalfa
Effect of pre-wetting
Prewetted20 ± 1.870 ±2.555 ± 2.1
Not prewetted15 ± 7.572 ± 7.549 ± 3.2
Effect of sample size, mg
150 mg19.6 ± 5.383.9 ± 1.562.2 ± 0.4
250 mg18.5 ± 3.581.6 ± 0.458.6 ± 0.5
350 mg18.8 ± 3.977.1 ± 1.757.6 ± 1.6

The fineness of the grinding screen commonly used (1 mm sreen) is perfectly suitable and it seems useless to reduce further the fineness of the particles. McLeod and Minson (1978) have reported an increase from 2.7 to 3.3 in residual standard error if the fineness of the grinding passes from 1 to 0.4 mm.

Pre-wetting of the sample prior to incubation can play an important role, especially in the case of substrates with low digestibility because it increases digestibility and reduces the standard error (Table 3).

Procedures: The general scheme of “in vitro” fermentation methods that is proposed is presented in Figure 2.

Enzyme solubility techniques

The in vitro rumen fermentation method of Tilley and Terry (1963) has been widely used all over the world and has revealed itself as the most accurate for predicting the “in vivo” digestibility of forages in both temperate and tropical areas (Barnes 1973; McLeod and Minson 1969, 1976).

Procedures: The general scheme of “in vitro” fermentation methods that is proposed is presented in Figure 2.

Figure 2

Figure 2: General flow chart of the “in vitro” fermentation methods

Nevertheless, this method is very costly and depends on the availability of fresh rumen juice with constant activity.

The cellulolytic activity is one of the principal characteristics of the microbial population of the rumen. Donefer et al (1963) and Jarrige and Thivend (1969) have attempted to reproduce it by using the cellulase enzyme preparations extracted from fungi. These enzymes, currently called cellulase, come most frequently from Trichoderma viride or from Aspergillus niger. The first have cellulolytic, hemicellulolytic and proteolytic activities (McQueen and Van Soest 1975); moreover, they have the two components C1 and Cx, allowing them to decompose intact fibres (crystalline and amorphous zones).

Enzyme solubility techniques have several sources of variation: enzyme source, experimental conditions, composition and pH of the buffer solution.

Enzyme source: The commercial brands of enzyme are not pure and their activities can vary considerably (Table 4). This variation is inherent to their nature because they have often cellulolytic, hemicellulolytic and even proteolytic activities. Variations in one or the other of these activities has its influence on the overall result. This implies a need for determining previously the activity of the “cellulase” to be used either by using pure substrate (cellulose) or by using as substrate the product to be tested. The technique has been described by Mandels and Weber (1969).

TABLE 4 Cellulase solubility of NaOH treated and untreated straw (1.0 g of sample) when incubated with different brands of enzyme (0.75 g of cellulase for 48 hours) (Rexen 1977)
Brands of cellulaseUntreated strawNaOH treated straw
Organic soluble matter % DMRelative percentageOrganic soluble matter % DMRelative percentage
Onozuka ss (1)24.310050.9100
B (2)13.05432.965
BDH (3)23.111654.5107
Merck (4)23.49641.281

(1) Onozuka ss, Lot 223027 (Yakult Biochemicals Co Ltd, Japan)

(2) B (Danish Brands, Grindsted Factory)

(3) BDH (British Drug Houses Chemicals, Product n° 39074)

(4) Merck (Art. 2329)

Experimental conditions (temperature, incubation time): Generally, the temperatures used vary from 30 to 50°C (Jones and Hayward 1975; McQueen and Van Soest 1975; Goto and Minson 1977; Rexen 1977; McLeod and Minson 1978). Jones and Hayward (1973) have shown that there are no significant differences between 40 and 47°C and that the solubility of the dry matter decreases if the temperature exceeds 50°C. Rexen (1977) has concluded that there is no difference between 35 and 50°C; for practical reasons he proposes 40°C instead of 50°C with an allowance of ± 5°C without any effect on the results. McLeod and Minson (1978), on the contrary, reported an important role of temperature with feeds of low digestibility. Thus, they obtained 46.5 and 50% solubilities of the dry matter for incubation temperatures of 39 and 50°C respectively. An increase in temperature did not improve the accuracy of the prediction.

According to the literature, incubation time varies on average from 24 to 48 h (Rexen 1977; McLeod and Minson 1978). The few data available seem to indicate that it might be necessary to increase incubation time (48 h and more) for the poor feeds.

Figure 3

Figure 3:
Gerneral flow chart of “Cellulase” solubility methods

pH and composition of the buffer solution: It is generally accepted that an acetate buffer solution (0.05 to 0.1 M) and a pH of 4.6 are suitable for the enzyme solubility measurements (McQueen and Van Soest 1975; Goto and Minson 1977; McLeod and Minson 1978). Rexen (1977) compared two types of buffer solutions, namely acetate and citrate at different molarities (0.1 to 1.0M), and several types of enzyme brands, to solubilize the dry matter of straw treated or untreated with sodium hydroxide. He concluded that 0.1 M citrate buffer was more suitable because this maintains the pH constant throughout the incubation phase, thus avoiding the readjustment of the pH even for 8% NaOH treated straw.

Enzyme/substrate ratio: The choice of the optimum concentration of “cellulase” to be used depends on the nature of the substrate (potential solubility) and on the origin of the commercial brand of enzyme. In practice, it is necessary to determine beforehand, for each kind of substrate, an optimum concentration for a given enzyme. Standardization of this technique is highly recommended.

Thus an enzyme/substrate ratio of 1.125 of Onozuka ss is more efficient than the ratios 17 or 12 for “cellulase” and hemicellulases” of Aspergillus niger (McQueen and Van Soest 1975). Rexen (1977) used straw as substrate and three enzyme brands (BDH, Onozuka and D). There was no difference between 0.5 and 1.5 of “cellulase” for 1 g of substrate. McLeod and Minson (1978) reported that the enzyme concentration improves significantly the residual standard error of the regressions related to in vivo data (Table 5). However, higher enzyme concentrations could be envisaged for low digestibility feeds.

TABLE 5 Effect of cellulase concentration and duration of incubation at 39°C upon the RSD (% Units) of regressions relating DMD in vivo to DMS in pepsin-cellulase of 5 grasses and 5 legumes species (Mc Leod and Minson 1978)
 Number of samplesPepsin - cellulase method
Incubation time (hr)484824
Cellulase concentration (g/100ml)2.50.6252.5
All grasses50±2.4±2.7±2.7
All legumes32±3.0±3.4±2.6
All species82±2.8±3.1±3.0

The general scheme of “cellulase” solubility techniques that is proposed is presented in Figure 3.


The majority of authors agree that the “in vitro” fermentation technique with rumen juice of Tilley and Terry (1963) offers the best laboratory estimation of “in vivo” digestibility (Oh et al 1966; Deinum and Van Soest 1969; McLeod and Minson 1969, 76, 78; Barnes 1973; Scales et al 1974; Schmid et al 1975; Terry et al 1978). Others hold that the enzyme solubility methods are equivalent, if not more accurate, than “in vitro” digestibility (Donefer et al 1963; Jones and Hayward 1973; Jarrige et al 1970; McLeod and Minson 1980; Demarquilly and Jarrige 1981).

TABLE 6 Correlations (r) and residual standard deviations (RSD) between in vivo DMD and in vitro rumen fermentation and enzyme solubility of DM
 No of samplesIn vitro rumen fermentationEnzymesolubility
Mc Leod & Minson 197850-2.100.942.60
Mellenberger et al 1970-0.90---
Aerts et al 1975–1976420.922.21--
Deinum & Van Soest 1969120.861.73--
Oh et al 1966320.83---
Jones & Hayward 1973170.952.000.912.70
Johnson et al 1964220.88---
Goto & Minson 197745-2.100.942.70
McLeod & Minson 197832-2.700.913.10
Terry et al 1978250.961.910.873.17
Oh et al 1966240.97---
McQueen & Van Soest 1975180.95-0.80-
Mc Leod & Minson 197882-2.60-3.00
Scales et al 19746.0843.20--
Terry et al 197873.0971.600.823.80
Van der Koelken & Van Es 1973560.882.96--
Hersherberger et al 1959110.77---
Hersherberger et al 1959350.97---
Chenost 1970680.754.71--
Cottyn et al 1970250.893.42--
Cottyn et al 1970940.812.18--
Johnson et al 1964260.78---
Johnson et al 1964650.72---
Scales et al 1974240.862.40--
Baumgardt et al 1962270.783.41--
Asplund et al 1958110.71---
Clark & Mott 196060.82---
Chenost 1970570.872.75--
Cottyn et al 1970310.930.74--
Marten et al 1975250.841.78--
Marten et al 1975260.921.90--
Aerts et al 1975–1976560.953.43--
Cottyn et al 1970250.622.96--
Mellenberger et al 1970-0.97---
Oh et al 1966210.97---
Johnson et al 1964170.44---
Giger et al 1979360.94---
Rexen 1977500.94-0.913.58

From the comparative data given in Table 6, it would seem that the enzyme solubility method gives rise to slightly lower correlation coefficients and to higher residual standard errors than the “in vitro” digestibility technique. In all the cases, the digestibility values obtained by enzyme solubility are lower than those obtained with rumen juice (Barnes et al 1964). Prior application of pepsin-HCL solubilisation improves this situation, ensuring at the same time a better precision (Jones and Hayward 1975; McQueen and Van Soest 1975). Pre-extraction with neutral detergent increases the amount of fibre degraded by cellulase (Roughan and Holland 1977; Dowman and Collins 1982). Van Soest et al (1966) reported that utilization of the digestion of the cell wall fraction (NDF) predicts more accurately “in vivo” digestibility than the digestibility of the dry matter. Scales et al (1974) and Oh et al (1966) affirm the contrary, whereas Meyer et al (1971) claimed that the two procedures were similar.

TABLE 7 Correlation coefficients (r) between in vitro determinations and intake, relative intake, energy digestibility and nutritive value index
 DM intakeRelative intake*Energy digestibilityNutritive value index**
Tilley and Terry DMD (1)0.21-0.900.57
One stage TT DMD (1)0.05-0.390.05
In vitro digested NDF (1)0.22-0.770.43
In vitro cellulose digestion (2)----0.78--
In vitro DMD (3)----0.92--
24 hrs in vitro DMD (4)0.76------
12 hrs in vitro cellulose dig. (5)--0.770.740.86
12 hrs in vitro cellulose dig (6)--0.830.870.91
In vitro cellulose dig. (7)--0.500.760.71
Cellulase solubility (5)--0.850.680.92
Cellulase pepsin solubility(5)--0.860.700.93

(1) Meyer et al 1971

(2) Baumgardt et al 1962

(3) Hersherberger et al 1959

(4) Chenost 1970

(5) Donefer et al 1963

(6) Donefer et al 1960

(7) Johnson et al 1964

* Relative intake = observed intake × 100/80 (w° .75)

** Nutritive value index = relative intake × % energy digestibility.

Prediction of intake

Generally speaking, “in vitro” techniques are not suitable for predicting the voluntary intake. Some studies have indicated limited success when intake was correlated with 6 or 12 h “in vitro” digestibility of dry matter (Chenost 1970) or with digestion of cellulose by rumen juice or “cellulase” (Donefer et al 1960, 1963). For temperate species, voluntary intake is better correlated with the cell wall content, whereas for tropical species a better index might be the resistance to grinding (McLeod and Minson 1972).


The apparently indigestible fraction of the cell content, considered as an entity or indigestible cytoplasm, varies little; on average, it represents 12 to 14% of the dry matter of forage according to the concordant results of several authors (Van Soest 1967; Deinum 1971; Osbourn et al 1976; Jarrige 1980). The apparently indigestible organic matter of forages depends closely on the apparently indigestible fraction of the cell walls. Predicting the digestibility of forages amounts to predicting the percentage of their non digestible cell-wall.

Among the existing chemical procedures for determining this fraction, Van Soest's method is the most commonly utilized and it is found to be suitable at least for temperate forages. The precision of the method can be slightly enhanced by prolonging the extraction time to 2 h or more, or by increasing the concentration of the acid solution (McLeod and Minson 1972).

Can the method of Van Soest be applied to all forages and to tropical crop residues and by-products? It is difficult to give a positive answer a priori and a certain adaptation in the method would seem to be necessary, especially for the non-conventional agro-industrial by-products. Thus, for example, the cell wall content of olive cake is 55% of the DM, whereas the in vivo digestibility of the DM is only 30 to 35% (Nefzaoui et al 1982, 1983). In vivo digestibility of sugar cane bagasse is 25 to 27%, while the summative equation of Van Soest predicts 38% (Molina, personal communication).

The trials made with olive cakes seem to indicate that 1 h extraction with the acid detergent solution is not sufficient for all products. While the NDF is stabilized after 1 h of extraction, the ADF goes on diminishing till 8 h and probably beyond (Figure 4). Likewise, the lignin (72% H2SO4) on the NDF residue seems to be stabilized after 2 h with a rate similar to that of the NDF. On the contrary, the lignin values determined on the ADF residue are lower and diminish if the extraction time increases. This point seems to indicate that a part of the lignin or the complete tannin protein (J Reed, personal communication) may have been solubilized during the extraction. Measurement of the optical density at 280 nm (OD 280 nm) of the extraction juice also confirms it (Figure 4). The OD 280 nm curve is a reversed image of that of the lignin.

The acid detergent fibre insoluble nitrogen (ADIN) also diminishes if the extraction time increases; but it explains only partially the reduction of the ADF. These two remarks do not exclude the solubilization of the cellulose or of a part of the lignin during the first hour of extraction. Only further investigations can provide some coherent answers. However the preliminary experiments pose clearly the problem of optimum extraction time appropriate to non-conventional by-products.

We think that the extraction with neutral detergent solution, prior to the determination of lignin, is more uniform and is more suitable than that with acid detergent solution; and that the determination of NDF does not seem to pose problems. On the contrary, problems of ADF and ADL analysis may lead to errors in the use of the summative equations for predicting nutritive value from the chemical composition of the cell wall. The OD at 280 nm reflects certainly the solubilization of a part of the lignin. Thus, the absorption measurement at 280 nm of the filtrate from alkali-treated olive cake after extraction (1 h) by refluxing with distilled water, is strongly correlated with the ADL content (Figure 2).

Figure 4

Figure 4:
Yields of NDF, ADF, and ADL from samples of olive cakes hydrolysed for different times and the corresponding optical density at 280 nm.

NDF (*—), (*—),ADL after neutral detergent extraction (•—),, ADL after acid detergent extraction (•—),, OD 280 nm on soluable NDS (*---) and OD 280 nm on Soluable ADS (*—)

The regressions show:

The OD 280 nm can be considered in this case to be as efficient for predicting the digestibility as the ADL content, or other components of the cell wall (Table 8).

TABLE 8 Simple correlations (r) and residual standard deviations between in sacco organic matter digestibility and lignin and optical density of alkali treated olive cake (Mefzaoui & Vanbelle, unpublished)
 In sacco OMD %ADLnc * % DMADLc** % DMOD 280 nm
In sacco OMD,%--0.943.260.904.230.904.24
ADLnc% DM  --0.990.870.871.48
ADLc% DM    --0.811.55

* nc: not corrected for N bound in ADF;

** c: corrected for N bound in ADF

Figure 5

Figure 5:
Graphs showing regressions relating lignin to OD 280 nm and “in sacco” OMD to lignin and OD 280 nm. Untreated (bull), NaOH treated (bull), Na2CO3 (bull), and ammonia treated (bull) olive cakes (Nefzaui and varibelle unpublished)

The question now is whether this relation is valid for other by-products or for other kinds of treatments involving high temperatures (heat damage - Maillard reactions)?


Of all the methods that have been discussed, which are to be chosen? The in vitro fermentation with rumen juice or enzyme solubility? The “in vitro” digestibility technique of Tilley and Terry is certainly the best one available at present for predicting the nutritive value of forages and by-products in spite of some obvious disadvantages:

Enzyme techniques, despite a slight lower precision, offer several advantages:

The major disadvantage is in obtaining enzyme brands of wide spectrum of action with constant activity from one batch to another.

Chemical and in vitro methods are indissociable and complementary. The adaptation of the fractional separation technique of Van Soest to non conventional crop residues and agro-industrial by-products constitutes a priority for discerning the problems relative to the understanding of the cell wall indigestible fraction.

General information about the environmental conditions (temperature, pluviometry, etc.) of the species, the state of development, can be more reliable than a simple chemical criterion.

In vitro determinations destined to predict the digestibility of feeds are not absolute techniques. Multiple factors are necessary to be able to modify the results obtained with these methods. To obtain less variable results among several laboratories it would be necessary:


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