In vitro quality assessment of tannin-containing
tropical shrub legumes: protein and fibre digestion
C.S. McSweeney
a,*, B. Palmer
b, R. Bunch
a, D.O. Krause
a aCSIRO Tropical Agriculture, Long Pocket Laboratories, Private Bag No. 3 PO,Indooroopilly, 4068 Qld., Australia
bCSIRO Tropical Agriculture, Davies Laboratory, Private Mail Bag, Aitkenvale,
Townsville, 4814 Qld., Australia
Received 29 March 1999; received in revised form 9 August 1999; accepted 26 August 1999
Abstract
In vitro techniques were evaluated to determine the nutritive value of a selection of tanniniferous tree and shrub legumes (Calliandra calothyrsus, Leucaena leucocephala L. diversifolia and L. pallida) compared with lucerne (Medicago sativa). Polyethylene glycol (PEG) was also added to some in vitro fermentations (10 mg PEG/50 mg plant substrate) to assess the effects of tannins on digestion of dry matter (DM), neutral detergent fibre (NDF) and nitrogen (N).
Total tannin content was poorly correlated with digestibility of dry matter and nitrogen. Apparent digestibility of dry matter and N were significantly different between plants and ranked in the following order; lucerne >L. leucocephala>L. diversifolia>L. pallida>C. calothyrsus. Ammo-nia was not produced (net accumulation) during 72 h fermentation ofC. calothyrsus,L. diversifolia
andL. pallidaalthough apparent nitrogen digestion in these plants ranged from 36.9 to 44.3%. Acid-pepsin digestion resulted in a further 17±22% of dry matter digestion in the shrub legumes compared with 8% in lucerne following 72 h fermentation. The amount of acid-pepsin digestible N available was lowest for lucerne (8.4%) and highest forL. pallida(38.9%) with the other legumes ranging from 26.5 to 36.8%.
The PEG addition caused a significant increase in rate and extent of DM and NDF digestibility and ammonia production for all the tannin containing shrub legumes but not for lucerne. However, DM loss and fermentability of these plants appeared to be poorly correlated because PEG addition resulted in an increase in volatile fatty acid production ranging from 3.7 to 202% compared with an increase in apparent DM digestibility of 9.1±30%.
It is concluded that in vitro evaluation of apparent DM and N digestibility of tannin containing plants provides a poor indication of true digestion (fermentability) and thus measurements of
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*Corresponding author. Tel.:61-7-3214-2820; fax:61-7-3214-2880
E-mail address: [email protected] (C.S. McSweeney)
fermentation end-products (ammonia and short and branched chain fatty acids) should also be undertaken to evaluate nutritive value. Also, addition of PEG to in vitro fermentations can be used to determine the effect of tannins on N digestibility. Based on the in vitro methods of rumen fermentation used in this study, nutritive value of the tanniniferous forages is ranked as follows:L. leucocephala>L. diversifoliaandL. pallida>C. calothyrsus.#1999 Elsevier Science B.V. All rights reserved.
Keywords: Tannins; Rumen; Polyethylene glycol; Tropical legumes; Nutritive value
1. Introduction
A major limitation to ruminant production in dry tropical regions is poor nutrition. Annual growth rates of animals are restricted by low nitrogen and high fibre content of native grasses and crop residues which form the basis of the diets in these climatic zones. Supplementation of tropical roughages with leguminous fodder trees and shrubs (FTS) is a promising way of alleviating nitrogen deficiencies.
Selecting plants for introduction usually involves ranking related accessions on agronomic potential and nutritive value. Laboratory techniques are often used to assess nutritive value and thus predict animal performance. The nylon bag technique (Mehrez and Orskov, 1977) is used routinely to determine digestibility of substrate in the rumen while in vitro fermentation techniques that measure gas production (Menke et al., 1979) and apparent digestibility (Tilley and Terry, 1963) are also used to determine nutritive value. However, FTS often contain anti-nutritive factors such as condensed tannins which affect their nutritive value (Ahn et al., 1989). Apparent digestibility can be confounded by the fact that soluble complexes form between condensed tannin, protein and carbohydrate. It has been suggested that the gas pro-duction technique is more reliable than the nylon bag method for determining nutritive value of feed containing anti-nutritive factors (Khazaal et al., 1993). However, the two techniques are poorly correlated when determining the nutritive value of tropical legumes of lower digestibility (Siaw et al., 1993). Furthermore, the gas production technique is often employed to determine digestibility and infer nutritive value without assessing nitrogen degradability even though tropical legumes are commonly used in ruminant production systems to correct a primary nitrogen deficiency (Siaw et al., 1993). Recent studies have also shown that the addition of polyethylene glycol (PEG) or polyvinylpyrrolidone (PVP) to in vitro fermentations of tannin containing forages will improve dry matter digestibility and thus provides insight into the nutritional significance of the tannins (Makkar et al., 1995; Khazaal et al., 1996). Curiously, the use of PEG or PVP to assess effect of tannins on in vitro N digestibility has received little attention considering tanniniferous legumes are usually fed as nitrogen supplements.
2. Materials and methods
2.1. Plant samples and preparation
The shrub legumes Calliandra calothyrsus CPI 115690, Leucaena leucocephala cv Cunningham,L. diversifoliaCPI 33820 andL. pallidaCQ 3439 were grown and harvested at Lansdown Research Station, 50 km south of Townsville, Queensland, Australia (198400
S, 1468510
E). The area was periodically grazed and then slashed to about 50 cm and allowed to regrow to the next grazing period. After a period of regrowth, the first five fully expanded leaves were collected as the standard sample from each plot. These tropical shrub legumes were compared with the temperate legumeMedicago sativa(lucerne). Adequate quantities of samples were collected (approximately 1 kg) from each plant and oven dried at 658C in a force draught oven for 48 h. Dried samples were ground in a Wiley mill fitted with a 1 mm screen and stored in air-tight containers atÿ708C until required. Temperature of drying and method of drying (e.g., oven drying versus freeze drying) will affect digestibility characteristics of tanniniferous forages but rank order of digestibility between different plants is usually unaffected by drying technique (Palmer, unpublished data).
2.2. Anaerobic techniques and media
The anaerobic techniques of Hungate (1969) as modified by Bryant (1972) were used for the growth of organisms and preparation of media. Finely milled oven-dried (658C) plant material (50 mg) was weighed into sterile Balch tubes and autoclaved basal medium (10 ml) was then dispensed under sterile conditions into the Balch tubes and immediately stopped. Basal medium contained 15% (v/v) clarified rumen fluid, 20% (v/v) macro mineral Solution A and 0.01% (v/v) micro mineral Solution B (Menke et al., 1979), 0.78% (w/v) NaHCO3, 0.25% (w/v) yeast extract, 0.03% (w/v) Na2S9H2O and 0.001% (w/v) resazurin, (Caldwell and Bryant, 1966), 0.0001% (w/v) hemin and NH4Cl (3 mM). Basal medium (pH 6.7) contained less than 0.5 mM ammonia N prior to addition of NH4Cl. Polyethylene glycol (PEG, MW 4000; BDH Cat No. 29576 HM) was also added in solution to culture tubes (10 mg/10 ml basal medium) as a treatment to counteract the effect of tannins and thus determine the potential nutritive value of the plant if tannins were not present. Fermentations were done in triplicate and uninoculated controls, with and without PEG were routinely included. B-vitamins (Lowe et al., 1985) were added to each tube of medium just prior to inoculation and incubations were at 398C.
2.3. In vitro digestibility
and neutral detergent fibre (NDF) digestibility were made from tubes sacrificed at varying intervals during a period of 72 h after inoculation. Percentage DM loss at 0 h represents the amount of DM that is soluble in uninoculated media. Tubes were also sub-sampled for measurements of ammonia production as an indicator of the rate of protein digestion.
In a separate experiment, the two stage technique (Tilley and Terry, 1963) for in vitro digestion of forage plants was used to determine potential digestibility of dry matter, and nitrogen and short chain fatty acid production, as well as acid-pepsin digestible protein that remained following fermentation for 72 h. The methods were essentially the same as those described by Tilley and Terry (1963) except approximately 0.5 g of plant material was used as substrate and the medium, inoculum size and ratio of plant material to culture fluid were as described above. Triplicate assays were performed at each time point and measurements corrected for changes in uninoculated controls. Short chain fatty acid production was not measured for lucerne.
2.4. Chemical analyses
The indophenol method for the determination of ammonia as described by Chaney and Marbach (1962) was used to estimate the rate and amount of ammonia production in cultures. Volatile fatty acids (VFA) in culture fluid were analysed by high performance liquid chromatography using a Waters System (Waters, Milford, Mass., 01757) equipped with an Aminex HPX-87 Cation exchange column (300 mm7.8 mm) for organic acids and a microguard column (Bio-Rad Laboratories, Hercules, CA, 94547) with a column heater (Waters Model 1122/WTC-120). Organic acids and ethanol were eluted using a mobile phase of 2.5% acetonitirile in 0.2% v/v phosphoric acid at a flow rate of 0.7 ml/ min., a column temperature of 608C and UV detection at 210 nm. NDF and ADF of plant substrates was determined by the method of Van Soest et al. (1991) using the micro analysis technique of Pell and Schofield (1993). Total condensed tannin was determined by the butanol-HCl method (Terrill et al., 1992). Nitrogen content of samples was measured using the Kjeldahl method (AOAC, 1980).
2.5. Statistical analysis
Statistical analysis of dry matter disappearance, ammonia production and N digestion was by analysis of variance with differences determined by the method of least significant difference at the 5% level (P< 0.05). A repeated measures design was used to determine the effect of PEG treatment on digestion and ammonia production during 72 h incubations of substrate where a series of measurements were made during the course of the incubation. All statistical analyses were done with Statistica 5.0 software (StatSoft, Inc., Tulsa, OK., 74104).
3. Results
significantly higher NDF content (P< 0.05) than the shrub legumes. Nitrogen content of the five plants was relatively high and ranged from 2.7 to 3.6% DM. Nitrogen associated with the NDF fraction appeared to be higher in the shrub legumes but this may have be due to association of protein and tannin with NDF when the plant samples were originally dried. Total condensed tannin concentrations in calliandra and the leucaenas ranged from 3.8 to 7.7% DM with greater than 84% of the tannin in a free form.
3.1. Apparent digestion of nitrogen and fermentation end-products
There were significant differences (P< 0.05) in digestion of nitrogen between the tropical legumes tested but these were not correlated with tannin content (Table 2). Lucerne had significantly higher water soluble N than the legumes containing tannin and L. leucocephala had the lowest soluble N content. Apparent microbial digestion of nitrogen was highest for lucerne followed by the shrub legumes in the following order; Table 1
Neutral detergent fibre (NDF), acid detergent fibre (ADF), nitrogen (N) and condensed tannin content (%) of lucerne,Calliandra calothyrsus,Leucaena leucocephala,L. diversifoliaandL. pallidaa
Lucerne Calliandra L. leucocephala L. diversifolia L. pallida Pooled sem
NDF (% DM) 41.4 a 37.6 b 27.8 e 29.4 d 34.3 c 1.1
ADF (% DM) 28.4 a 22.7 b 14.7 e 17.5 d 19.6 c 1.3
N (% DM)b 3.17 3.64 2.72 3.48 3.31 ±
N (% NDF)b 1.12 3.68 2.86 3.21 2.16 ±
Condensed tannin (% DM)
Free n.t. 4.78 b 3.32 c 6.57 a 4.00 c 0.46
Total n.t. 5.71 b 3.84 d 7.70 a 4.72 c 0.55
aValues within rows with different letters differ significantly (P< 0.05). bNot statistically tested due to lack of replication.
Abbreviations: DM, dry matter; n.t., not tested.
Table 2
Nitrogen digestion from lucerne,Calliandra calothyrsus,Leucaena leucocephala,L. diversifoliaand incubated with rumen fluid (72 h) and followed by acid-pepsin treatmenta
Lucerne Calliandra L. leucocephala L. diversifolia L. pallida
N incubated (mg)b 14.47 (0.s01) 16.52 (0.07) 12.45 (0.11) 15.87 (0.03) 15.16 (0.02) Water soluble N (% total)c 47.9 (0.1) a 20.9 (1.6) b 15.3 (1.3) c 22.3 (0.1) b 21.3 (0.1) b Microbial digested N (% total)d 78.3 (0.7) a 36.9 (0.1) e 49.3 (0.1) b 44.3 (0.1) c 40.1 (0.1) d Acid pepsin digested N (% total)e 8.4 (0.8) a 36.8 (0.6) c 26.5 (0.4) b 36.1 (0.7) c 38.9 (0.6)d Digestible N (% total)f 86.7 (0.1) a 73.7 (0.3) e 75.8 (0.4) c 80.4 (0.2) b 78.9 (0.0) d
aValues within rows with different letters differ significantly (P< 0.05) and values in parenthesis are the standard error of the mean.
bmg N/500 mg plant substrate.
cN solubilized in uninoculated control tubes.
dN apparently digested during fermentation including water soluble N. eN apparently digested by acid pepsin treatment following microbial digestion.
L. leucocephala>L. diversifolia>L. pallida>C. calothyrsus. The amount of acid pepsin digestible N available following a 72 h fermentation was lowest for lucerne (8.4%) and highest forL. pallida(38.9%) with the other legumes ranging from 26.5 to 36.8%. Apparent digestible N was highest for lucerne (86.7%) although the majority of total N (73.7±80.4%) was apparently available for digestion in the shrub legumes even though they contained substantial amounts of condensed tannin.
Ammonia was not apparently produced but rather consumed during 72 h fermentations of C. calothyrsus, L. diversifolia and L. pallida (Table 3). Accumulation of ammonia occurred in fermentations of lucerne (11.5 mM) andL. leucocephala(5.44 mM) which both had the highest apparent microbial digestion of nitrogen.
Volatile fatty acid (VFA) concentrations in uninoculated cultures were negligible (data not shown). Production of VFA were significantly reduced by the presence of tannin in some of the legumes (Table 4). Inclusion of PEG in fermentations of the tanniniferous plants resulted in a significant increase in volatile acid production with all plants except L. leucocephalawhich did not change significantly. The increase in VFA production for calliandra,L. diversifoliaandL. pallidawere 202, 37 and 129%, respectively. The PEG addition caused a significant increase in acetate to propionate ratio only with L. diversifolia. Branched chain volatile fatty acids (BCVFA) were not produced from fermentations of calliandra, L. diversifolia and L. pallida without PEG but BCVFA increased significantly (P< 0.05) with all tanniniferous plants in the presence of PEG. The highest concentrations (P< 0.05) of BCVFA occurred with PEG additions to L. diversifoliaandL. pallida.
3.2. Apparent digestion of dry matter
Apparent dry matter digestion was significantly different between legume species (Table 3). A large proportion of the dry matter (approximately 0.3) was solubilized immediately from plant material in uninoculated cultures (Table 3). Apparent dry matter digestion was highest for lucerne (63.1%) whileL. leucocephala(51%) had the highest digestibility of the tannin containing legumes. Acid/pepsin digestion resulted in digestion
Table 3
Dry matter loss and ammonia production from lucerne,Calliandra calothyrsus,Leucaena leucocephala,L. diversifolia L. pallidaincubated (72 h) with rumen fluida
Lucerne Calliandra L. leucocephala L. diversifolia L. pallida
DM loss (%)
Uninoculated 36.1 (0.6) b 28.9 (0.6) d 38.9 (0.5) a 30.7 (0.5) c 31.2 (0.2) c Inoculated 63.1 (0.4) a 32.2 (1.2) e 51.0 (0.1) b 43.3 (1.0) c 38.5 (0.9) d Acid/pepsin digest 71.1 (0.2) a 53.8 (0.2) e 68.8 (0.3) b 60.4 (0.8) c 56.7 (0.4) d
Ammonia (mM)
Uninoculated 4.46 (0.03) b 3.33 (0.03) d 3.83 (0.05) c 4.70 (0.09) a 4.77 (0.11) a Inoculated 11.06 (0.06) a 2.86 (0.04) c 5.44 (0.16) b 2.18 (0.09) e 2.50 (0.12) d
Table 4
End products of fermentation fromCalliandra calothyrsus,Leucaena leucocephala,L. diversifoliaandL. pallidaincubated with rumen fluid, plus or minus polyethylene glycol (PEG)a
Calliandra L. leucocephala L. diversifolia L. pallida
ÿPEG PEG ÿPEG PEG ÿPEG PEG ÿPEG PEG
Total VFA (mM) 6.96 a (0.07) 20.99 b (0.68) 25.41 c (0.46) 26.34 c (0.52) 12.57 e (0.19) 28.82 f (0.30) 19.23 d (1.09) 26.39 c (0.50) Acetate : propionate 2.59 a (0.14) 2.63 a (0.06) 2.15 b (0.01) 2.06 b (0.03) 2.05 b (0.02) 2.38 c (0.04) 2.0 b (0.05) 2.06 b (0.05) BCVFA (mM) 0 a 0.530 b (0.021) 0.253 c (0.021) 0.433 d (0.050) 0 a 0.753 e (0.028) 0 a 0.722 e (0.028)
aValues within rows with different letters differ significantly (P< 0.05) and values in parenthesis are the standard error of the mean. BCVFA, branched chain volatile fatty acids.
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of a further 17±22 units of dry matter in the shrub legumes compared with 8 units in lucerne (Table 3).
3.3. Kinetics of fermentation of protein and dry matter in lucerne and tannin containing legumes
Addition of PEG caused a significant and marked increase (P< 0.05) in the rate and extent of ammonia production for all tannin containing shrub legumes (Fig. 1). A period of about 20±30 h elapsed before there was an increase in rate and extent of ammonia production which represents the lag phase of growth of the small inoculum (0.1 ml/10 ml culture) used to initiate the fermentation. Significant amounts of ammonia accumulated in fermentations of L. leucocephala without PEG but ammonia was not apparently produced in fermentations ofL. pallida,L. diversifoliaandC. calothyrsusunder the same conditions. Ammonia concentration in uninoculated controls (with and without PEG) of shrub legumes varied less than 1 mM during 96 h of incubation (data not shown). Production of ammonia (approximately 4 mM) from lucerne was not significantly affected by PEG (Fig. 3). Ammonia concentration peaked earlier (52 versus 64±72 h; P< 0.05) for lucerne compared with the shrub legumes treated with PEG. Initially net ammonia production was negative forL. leucocephalabefore ammonia accumulated.
PEG treatment resulted in an increase in rate and extent of DM and NDF digestibility for all the tannin containing shrub legumes withL. leucocephalabeing least affected (Fig. 2). The ranking for DM matter and NDF digestibilities of the shrub legumes with PEG was C. calothyrsus<L. diversifolia, L. pallida<L. leucocephala(P< 0.05). Similarly, without PEG,C. calothyrsushad the lowest (P< 0.05) ranking for DM digestibility (C. calothyrsus<L. diversifolia<L. pallida<L. leucocephala) whileL leucocephalaranked highest (P< 0.05) for NDF digestibility (C. calothyrsus<L. diversifolia<L. pallida<L. leucocephala). Dry matter digestibility of lucerne was not significantly affected by PEG (Fig. 3). Lucerne had the highest DM digestibility (P< 0.05) of the legumes except forL. leucocephalaPEG which was not significantly different. It was calculated that 100% conversion of nitrogen in the shrub legumes to ammonia would have resulted in ammonia concentrations in the culture fluid (minus ammonia in media constituents) of calliandra (13.0 mM), L. diversifolia (12.4 mM), L. pallida (11.9 mM) and L. leucocephala (9.7 mM).
4. Discussion
Fig. 1. Ammonia production during in vitro fermentations of calliandra (open circle),L. leucocephala(solid circle),L. diversifolia(open square) andL. pallida(solid square) in the presence and absence of polyethylene glycol (PEG).
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Fig. 3. Dry matter (DM) digestion and ammonia production during in vitro fermentation of lucerne in the presence (solid circle) and absence (open circle) of polyethylene glycol (PEG).
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preferable to sterilize plant material prior to inoculation with rumen microorganisms for in vitro fermentation to remove the effect of saphrophytic bacteria but this would affect potential digestibility of organic matter and protein particularly in tannin containing plants. However, in the current study volatile fatty acid concentrations in uninoculated cultures were negligible thus indicating minimal growth of residual bacteria on unsterilized plant material.
The Tilley and Terry (1963) method and nylon bag technique measure apparent digestion of dry matter and nitrogen but cannot account for material that is solubilized or escapes from the nylon bag and is not digested. In conventional feeds this is probably of minor consequence but this study shows that true digestibility of tanniniferous forages is likely to be inaccurate based solely on measurements of apparent digestibility. In this study, there was a 1.7-fold difference in DM digestibility between the tanninferous plants but a 3.7-fold difference in VFA production which suggests that a proportion of the digested DM fraction may not be fermented in some tanniniferous forages. Also, the increase in both DM and NDF digestibility that occurred with PEG inclusion may have been underestimated since it has been shown that the addition of tannin-binding agents can reduce the true digestibility value by binding to NDF and thus being included as `artefact NDF and DM' (Makkar et al., 1995). These factors make it difficult to predict true digestibility of DM when tannins are present. The poor correlation between DM loss and fermentability of these plants is also demonstrated by the fact that VFA increased by 3.7±202% (Table 4) in response to PEG compared with apparent increases in DM digestibility of 9.1±30.0% (Fig. 2). This suggests that the response in digestibility to PEG is underestimated and that estimates of short chain volatile fatty acid (SCVFA) production may provide a better indication of fermentable carbohydrate. Furthermore, fermentation pattern (molar proportions of SCVFA) did not change substantially in response to PEG except for an increase in BCVFA which is indicative of increased fermentation of protein.
Tilley and Terry (1963) showed lower potential N digestibilities (74±80%) for all tannin containing shrub legumes. However, a large portion of this digestible N (35±50%) was only available following the acid pepsin digest compared with 9.7% for lucerne.
The acid pepsin digestible protein could be regarded as potential rumen by-pass protein but a limitation of this in vitro technique is that the fate of protein in the intestines following abomasal (acid-pepsin) digestion cannot be predicted. A recent report indicates that dietary protein complexed with tannin is made available in the abomasum and digested in the intestines but tannin released from protein-tannin complexes may react with non-dietary protein as it passes along the intestines thus counteracting the benefits of digestion of by-pass dietary protein (McNeill et al., 1999). The action of different plant tannins on digestion post-ruminally is, therefore, of critical importance in determining the potential nutritive value of the legume. L. leucocephala should probably be used as a standard for tanniniferous tree legumes with desirable nutritive value and fermentation characteristic. In the present study, rate and extent of N and DM digestion of L. leucocephalawas equivalent to lucerne when the tannin effect was removed and tannins seemed to have less influence on digestion in this legume than the others tested. The high nutritive value ofL. leucocephalais demonstrated by the exceptional liveweight gains of 1.0 kg/day in cattle grazing the plant as part of native pasture system in tropical Australia (Shelton and Jones, 1995).
Rates of degradation of DM and evolution of ammonia were measured to predict the synchrony of release of energy and N in the rumen. When there is a balance between protein and carbohydrate fermentation, ammonia and peptide N are incorporated more efficiently into microbial protein and less ammonia accumulates. However, in this study, energy (DM and NDF) appeared to be digested sooner than protein (ammonia evolution) for all legumes and, thus, it is unlikely that synchrony of carbohydrate and protein digestion had much influence on the extent of ammonia accumulation for each legume. Therefore, ammonia production was probably a reliable indicator of rate and extent of protein fermentation. Protein, in all tannin containing plants, was fermented to ammonia at a slower rate than lucerne. This appears to be a desirable attribute ofL. leucocephala since excessive fermentation of protein would be reduced while adequate amounts of ammonia (5 mM) would be available for microbial growth by cellulolytic bacteria. However, rate and extent of protein digestion in the other tanniniferous legumes was severely restricted by tannin which could reduce ruminal ammonia concentration to levels (<3 mM) which could limit microbial protein synthesis in the rumen (see Morrison and Mackie, 1996). Sheep fed a sole diet of wilted calliandra had ruminal ammonia concentrations below 3 mM (Palmer and co-workers, unpublished data).
Based on the in vitro methods of rumen fermentation used in this study, nutritive value of the tanniniferous forages is ranked as follows:L. leucocephala>L. diversifoliaandL. pallida>C. calothyrsus. However, the effect of tannins on in vitro digestibility can be affected by plant preservation methods such as freeze or oven drying (Ahn et al., 1989; Siaw et al., 1993; Balogun et al., 1998) and thus nutritive value of preserved material may not accurately represent fresh forages consumed by grazing ruminants. Therefore, the techniques described in this paper should be adapted for studying the kinetics of fermentation of fresh tanniniferous forages as this may provide a better indication of their true nutritive value.
Acknowledgements
This work was partly supported by the Australian Centre for International Agricultural Research (ACIAR).
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