Short communication
An approach to screening potential pasture
species for condensed tannin activity
R.J. Jones
a,*, J.H.F. Meyer
b, M. Bechaz
b, M.A. Stoltz
b aCSIRO Tropical Agriculture, Davies Laboratory, PMB Post Of®ce, Aitkenvale, Qld 4814, AustraliabARC-RFI, Private Bag X05, Lynn East, 0039, South Africa
Received 5 October 1999; received in revised form 19 January 2000; accepted 24 March 2000
Abstract
Freeze dried samples of the leaves of six tropical leguminous shrubs were studied. Digestibilities of dry matter (IVDMD) and nitrogen (IVND) were determined in vitro using rumen ¯uid from sheep in the presence and absence of polyethylene glycol 4000 (PEG).
The difference due to PEG, the tannin effect, was more marked for IVND than for IVDMD and varied with species. The improvement forAcacia boliviana,Calliandra calothyrsusandLeucaena trichandrawas large, intermediate withL. leucocephalaandL. pallidaand small withGliricidia sepium(range 5.5±33.8 digestibility units).
The tannin effect was poorly correlated with previously published (Jackson et al., 1996) data on the butanol CT levels in these samples: extractable CT (r20.0007); protein bound (r20.485); ®bre bound (r20.566); and total CT (r20.1473). The bound CT and total CT were negatively related to the PEG effect. Vanillin CT was positively correlated with the PEG effect (r20.578). Two species,A. bolivianaandC. calothyrsus,behaved differently to the others. Omitting these from the regressions improved the relationships with both butanol extractable CT and total butanol CT and with vanillin CT (r20.9).
For new pasture species, screening by using this modi®ed in vitro digestion technique would avoid the problems of using a known CT standard or of isolating CT standards for each species. It would also provide some information on digestibility.#2000 Published by Elsevier Science B.V.
Keywords:Tropical browse; PEG; In vitro digestion; Condensed tannins; PEG±tannin complexes 85 (2000) 269±277
*Corresponding author. Tel.:61-747538500; fax:61-747538600. E-mail address: [email protected] (R.J. Jones)
1. Introduction
Condensed tannins (CT) are known to have both positive and negative effects on nutritional quality of both herbaceous and shrub legumes. At low levels they can prevent bloat and improve by pass protein and some essential amino acids to the small intestine. At high levels the protein may be over protected and lost in the faeces, the activity of rumen bacteria reduced and the production of gut enzymes impaired (Barry, 1989; Kumar and D'Mello, 1995). For a range of tropical legumes extractable tannins were negatively correlated with nylon bag digestibility (Balogun et al., 1998).
Assessment of the CT effect is complicated for several reasons. The heterogeneous nature of CT and their instability, together with the lack of any satisfactory standard, make it dif®cult to assess them chemically (Hagerman and Butler, 1989). Furthermore, the concentration determined by any particular method may not re¯ect the activity of the CT in modifying nutritive value. The different reactivities from various species and possibly cultivars mean that there is no universal reference material. Using external reference standards can therefore lead to serious bias in assessing CT concentration. Obtaining internal standards is time consuming and costly and may require relatively large quantities of plant material. Such standards would be inappropriate for screening large numbers of samples from several genera. The alternative is to use a common standard, for example in New Zealand CT fromLotus pedunculatuswas used to screen a large number of tropical legumes (Jackson et al., 1996).
The proposed method for screening potential forage plants is based on the known ability of polyethylene glycol (PEG) to preferentially bind with tannins so preventing the formation of potentially indigestible tannin±protein complexes (Jones, 1965; Jones and Mangan, 1977). We measured in vitro digestibility by a modi®ed two stage Tilley and Terry (1963) method in which the residues were analysed for N to enable a N-digestion to be calculated. Samples were incubated in the presence and absence of PEG with the difference in N-digestibility as a measure of the CT activity in the samples. The objective of the study was to assess the differences in digestibility, especially N digestibility, between samples with and without PEG, and to relate these differences to the various measures of tannin content of the same samples.
2. Materials and methods
2.1. Plant samples
Six freeze dried tropical shrub legume leaf samples were used (Table 1). All had been harvested on the same morning from plants grown at the CSIRO Pasture Research Station at Lansdown, near Townsville, Queensland, Australia (198400S; 1468510E).
Samples were comprised of the terminal ®ve leaves on actively growing shoots. Aliquots of the samples had been analysed for CT by the vanillin/HCl and the butanol/HCl methods and for N by Kjeldahl digestion and the results published earlier (Jackson et al., 1996).
2.2. Digestion studies
In vitro digestion studies were made at the Agricultural Research Station, Roodeplaat, Lynn East near Pretoria, South Africa on 0.5 g samples of ground (to pass a 1 mm screen) forage.
The modi®ed Tilley and Terry (1963) method was that described by Jones and Barnes (1996) in which Stage 1 was extended to 72 h and Stage 2 reduced to 24 h. Rumen ¯uid from a sheep fed medium quality lucerne hay was the inoculum source. The PEG was added in solution to the samples in the tubes immediately before addition of the rumen ¯uid/buffer to provide 80 mg PEG/tube or 160 mg/g sample. Samples were incubated at 398C in a waterbath. The residues after digestion were dried, weighed and analysed for N (Kjeldahl and NIR spectroscopy). There were 6 forage species2 PEG treatments3 replicates.
Dry matter and N digestibilities were calculated from the initial and ®nal weights of sample DM and N, after corrections using data from the blank (no sample) tubes. The difference in IVND between the PEG treatments (the tannin effect) was related to the various CT values for these same samples. CT was extracted using acetone/water/diethyl ether and the concentration of extractable CT determined by the vanillin/HCl and butanol/ HCl methods. Protein-bound CT was measured in the residue from the acetone extractable fraction, after extraction in boiling 1% sodium dodecyl sulphate (SDS) containing 5% 2-b-mercaptoethanol, with butanol/HCl. Fibre-bound CT was measured by boiling the residue with butanol/HCl and SDS-2-b-mercaptoethanol. The CT standard used was extracted fromLotus pedunculatus. Details of the methods and the results for these samples have been published (Jackson et al., 1996).
2.3. Statistical analysis
A two-factor randomised block design with three replicates was used. The treatment effects on digestibility were analysed using ANOVA. Mean differences in IVND for the Table 1
The effect of PEG 4000 on the in vitro dry matter digestibility (IVDMD) of six tropical forage speciesa
Species Cultivar or CPIb IVDMD
ÿPEG PEG
Acacia boliviana 40175 46.2 49.3
Calliandra calothyrsus 115690 49.3 51.5
Gliricidia sepium 60796 75.5 75.3
Leucaena trichandra(diversifolia) 46568 57.9 59.7
Leucaena leucocephala cv. Cunningham 67.3 67.3
Leucaena pallida 84581 57.3 54.2
plus and minus PEG treatment for each species were regressed on the various measures of tannin content by standard regression techniques.
3. Results
In the absence of PEG, IVDMD ranged from 46.2% withA. bolivianato 75.5% with G. sepium. Within theLeucaenaspp., the range was 57.3% withL. pallidato 67.3% with L. leucocephala.
Overall, there was no signi®cant effect of PEG on IVDMD (p0.609). However, there was a large species effect (p<0.0001) and a signi®cant speciesPEG interaction (p0.0038). For two species,C. calothyrsus andA. boliviana,PEG increased IVDMD signi®cantly (p<0.05) (Table 1). With three species,L. leucocephala,L. trichandraand G. sepiumthere was little change, and withL. pallidathere was a signi®cant reduction (p<0.01) in IVDMD (Table 1).
There were large effects of both forage species and PEG on IVND (p<0.0001) and the interaction of speciesPEG was also highly signi®cant (p0.0001). PEG signi®cantly improved the IVND for all species (Table 2). The improvement varied from 5.6 digestibility units withG. sepiumto 33.8 digestibility units withA. boliviana(Table 2). For theLeucaena spp. the improvement ranged from 9.9 units withL. leucocephala to 15.8 units withL. trichandra.
In the absence of PEG, there was a range of IVND from 47.7% withC. calothyrsusto 80.2% withG. sepium. The range in the presence of PEG was much reduced: 79.8% with C. calothyrsusto 86.2% withL. leucocephala (Table 2).
The tannin effect was best related to the vanillin±HCl values, and very poorly related to the extractable butanol±HCl values (Fig. 1). The bound CT values of the butanol±HCl measures and the total butanol CT values were negatively related to the PEG effect (Fig. 2). Although the negative relations with the bound fractions may have been anticipated, the negative relation with total butanol±HCl CT was not (Fig. 2).
Table 2
The effect of PEG 4000 on the in vitro N digestibility (IVND) of six tropical forage speciesa
Species Treatment
ÿPEG PEG
Acacia boliviana 50.9 84.7
Calliandra calothyrsus 47.7 79.8
Gliricidia sepium 80.2 85.8
Leucaena trichandra(diversifolia) 67.9 83.7
Leucaena leucocephala 76.3 86.2
Leucaena pallida 68.1 79.9
aLSD for the speciesPEG interaction: 5% 1.68; 1% 2.29.
4. Discussion
Fig. 2. The relation between the IVND improvement due to PEG and the Butanol/HCl bound and total condensed tannins (CT). (A) Protein-bound CT; (B) Fibre-bound CT; (C) Total CT (extractableprotein-bound®bre-bound).
the digestion and subsequent hot water washing (Makkar et al., 1995). These complexes would then have the effect of reducing the estimated IVDMD. Previous work has shown that PEG±tannin complexes can lower the apparent and true digestibilities in in vitro studies (Makkar et al., 1995). In their work, the second stage of the Tilley and Terry (1963) digestion was replaced with an NDF determination and it is likely that the acid± pepsin treatment in our study may have removed more PEG±tannin complexes. However, as Makkar et al. (1995) point out, the presence of these PEG±tannin complexes in ADF fractions indicate that such `contamination' could occur in residues following the standard Tilley and Terry (1963) in vitro digestions. Use of labelled PEG (Silanikove et al., 1996) would quantify such contamination and enable true DM digestibility to be calculated. The variable results for IVDMD in response to PEG could well arise from the balance between favourable results due to improved digestibility on the one hand and negative effects due to insoluble tannin±PEG complexes in the residue on the other. No such problem occurs with the estimation of IVND since N input and N residue are readily measured.
The two shrub species showing positive IVDMD responses to PEG ÐC. calothyrsus andA. boliviana Ð also showed the largest responses to PEG in terms of IVND. The improvement in IVND with this latter species was 33.8 units (66%). This improvement in IVND in the presence of PEG can be explained by the removal of the protein binding action of CT. Removal of the CT inC. calothyrsusby extraction with 70% acetone also resulted in greater DMD and N losses (Whetton et al., 1997). They showed little improvement withG. sepiumwhich contains little or no free tannin. We obtained similar results using PEG to bind the tannins.
higher tannin levels than CPI 33820 (L.diversifolia) used in the grazing trial (Dalzell et al., 1998). The high digestibility and good animal perfomance onG. sepium is well established when no serious palatability problems occur (Bennison and Paterson, 1993). The results suggest that the method (involving the measurement of IVND as well as IVDMD in the Tilley and Terry (1963) technique in both the presence and absence of PEG 4000) will be useful for screening potential leguminous shrub species for tannin activity, especially where no standards are available. They also emphasise the fact that screening with standards that are not appropriate for the species concerned can lead to problems. For example, A. boliviana had the lowest level of total CT by the butanol/HCl assay and could have been considered a suitable species for further testing. However, it had the lowest N digestibility and the highest response to PEG of all the species tested.
Acknowledgements
One of us (RJJ) is indebted to the Australian Centre for International Agricultural Research (ACIAR) for funding to undertake a study in South Africa from which data presented in this paper was drawn. We also thank Patricia Ngwenya and Felicia Mokhele for their technical assistance in the laboratory and Mr. R.P. LeFeuvre for the statistical analyses.
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