Directory UMM :Data Elmu:jurnal:A:Animal Feed Science and Technology:Vol85.Issue3-4.Jun2000:
Animal Feed Science and Technology
85 (2000) 269±277
Short communication
An approach to screening potential pasture
species for condensed tannin activity
R.J. Jonesa,*, J.H.F. Meyerb, M. Bechazb, M.A. Stoltzb
a
CSIRO Tropical Agriculture, Davies Laboratory, PMB Post Of®ce, Aitkenvale, Qld 4814, Australia
b
ARC-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 for Acacia boliviana, Calliandra calothyrsus and Leucaena
trichandra was large, intermediate with L. leucocephala and L. pallida and small with Gliricidia
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. boliviana and C. 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
*
Corresponding author. Tel.: 61-747538500; fax: 61-747538600.
E-mail address: [email protected] (R.J. Jones)
0377-8401/00/$ ± see front matter # 2000 Published by Elsevier Science B.V.
PII: S 0 3 7 7 - 8 4 0 1 ( 0 0 ) 0 0 1 4 4 - 9
270
R.J. Jones et al. / Animal Feed Science and Technology 85 (2000) 269±277
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 from Lotus pedunculatus was 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 (198400 S; 1468510 E).
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).
271
R.J. Jones et al. / Animal Feed Science and Technology 85 (2000) 269±277
Table 1
The effect of PEG 4000 on the in vitro dry matter digestibility (IVDMD) of six tropical forage speciesa
Species
Acacia boliviana
Calliandra calothyrsus
Gliricidia sepium
Leucaena trichandra (diversifolia)
Leucaena leucocephala
Leucaena pallida
a
b
Cultivar or CPIb
40175
115690
60796
46568
cv. Cunningham
84581
IVDMD
ÿPEG
PEG
46.2
49.3
75.5
57.9
67.3
57.3
49.3
51.5
75.3
59.7
67.3
54.2
LSD for the speciesPEG interaction: 5% 2.16; 1% 2.94.
Commonwealth plant introduction number.
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 from Lotus 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
272
R.J. Jones et al. / Animal Feed Science and Technology 85 (2000) 269±277
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% with A. boliviana to 75.5% with
G. sepium. Within the Leucaena spp., the range was 57.3% with L. pallida to 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
85 (2000) 269±277
Short communication
An approach to screening potential pasture
species for condensed tannin activity
R.J. Jonesa,*, J.H.F. Meyerb, M. Bechazb, M.A. Stoltzb
a
CSIRO Tropical Agriculture, Davies Laboratory, PMB Post Of®ce, Aitkenvale, Qld 4814, Australia
b
ARC-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 for Acacia boliviana, Calliandra calothyrsus and Leucaena
trichandra was large, intermediate with L. leucocephala and L. pallida and small with Gliricidia
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. boliviana and C. 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
*
Corresponding author. Tel.: 61-747538500; fax: 61-747538600.
E-mail address: [email protected] (R.J. Jones)
0377-8401/00/$ ± see front matter # 2000 Published by Elsevier Science B.V.
PII: S 0 3 7 7 - 8 4 0 1 ( 0 0 ) 0 0 1 4 4 - 9
270
R.J. Jones et al. / Animal Feed Science and Technology 85 (2000) 269±277
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 from Lotus pedunculatus was 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 (198400 S; 1468510 E).
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).
271
R.J. Jones et al. / Animal Feed Science and Technology 85 (2000) 269±277
Table 1
The effect of PEG 4000 on the in vitro dry matter digestibility (IVDMD) of six tropical forage speciesa
Species
Acacia boliviana
Calliandra calothyrsus
Gliricidia sepium
Leucaena trichandra (diversifolia)
Leucaena leucocephala
Leucaena pallida
a
b
Cultivar or CPIb
40175
115690
60796
46568
cv. Cunningham
84581
IVDMD
ÿPEG
PEG
46.2
49.3
75.5
57.9
67.3
57.3
49.3
51.5
75.3
59.7
67.3
54.2
LSD for the speciesPEG interaction: 5% 2.16; 1% 2.94.
Commonwealth plant introduction number.
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 from Lotus 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
272
R.J. Jones et al. / Animal Feed Science and Technology 85 (2000) 269±277
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% with A. boliviana to 75.5% with
G. sepium. Within the Leucaena spp., the range was 57.3% with L. pallida to 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