Directory UMM :Data Elmu:jurnal:A:Animal Feed Science and Technology:Vol85.Issue3-4.Jun2000:
Animal Feed Science and Technology
85 (2000) 259±268
The effect of PEG addition in vitro on dry matter and
nitrogen digestibility of Calliandra calothyrsus and
Leucaena leucocephala leaf
B. Palmer, R.J. Jones*
CSIRO Tropical Agriculture, Davies Laboratory, PMB Post Of®ce, Aitkenvale, Qld 4814, Australia
Received 8 October 1999; received in revised form 24 January 2000; accepted 15 February 2000
Abstract
A modi®ed two stage in vitro digestion method was used to study the effects of rate of PEG addition
(0±1100 mg/g DM) on dry matter (IVDMD) and nitrogen digestibility (IVND) of freeze dried leaf
materialfrom actively growingshootsofCalliandra calothyrsus (Calliandra)and Leucaena leucocephala
cv. Cunningham (Leucaena). For both species, IVDMD and IVND increased asymptotically with PEG
rate; the IVND response being markedly greater for Calliandra. There was a three-fold greater amount
of PEG bound to the residue after Stage 1 (rumen ¯uid/buffer) digestion with Calliandra than with
Leucaena (105 versus 33 mg/g DM). The presence of PEG in the residue, which was not reduced after
Stage 2 (acid±pepsin) digestion, resulted in a higher residue weight and therefore an underestimated
IVDMD. Quanti®cation of the PEG in the residue enabled a corrected IVDMD (CIVDMD) to be estimated. PEG did not bias estimates of nitrogen digestibility in the same way. In a second experiment,
estimates of CIVDMD and IVND were made after Stage 1 and after Stage 2 in the presence and
absence of PEG at 160 mg/g sample. For Leucaena, there were small, but signi®cant, effects of PEG,
whereas with Calliandra there were large responses to PEG and to timing of addition. When measured
after Stage 1, IVND in the absence of PEG was not signi®cantly different to zero (compared with 57%
for Leucaena), whereas with PEG, IVND was 75% (compared with 68% for Leucaena). The low IVND
for Calliandra was associated with low NH4 N levels in the rumen ¯uid/buffer after Stage 1.
In summary, about 160 mg PEG/g sample is appropriate for most studies with tropical tanniniferous
shrub legumes to estimate any deleterious tannin effect. The use of PEG to estimate this effect on
IVDMD is not valid without accounting for the PEG bound to the residue. For estimation of the adverse
effect of tannins on IVND the use of PEG may be more appropriate. # 2000 Published by Elsevier
Science B.V.
Keywords: In vitro digestibility; Tannin containing shrub legumes; PEG binding; DMD; N digestibility
*
Corresponding author. Tel.: 61-7-47538520; fax: 61-7-47538600.
E-mail address: raymond.jones@tag.csiro.au (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 2 5 - 5
260
B. Palmer, R.J. Jones / Animal Feed Science and Technology 85 (2000) 259±268
1. Introduction
The ability of PEG to bind with condensed tannins (CT) has been used in ®eld and
laboratory studies to measure and to reduce the adverse effects of CT in ruminant diets
(Pritchard et al., 1988; Barry, 1989; Silanikove et al., 1994; McSweeney et al., 1999;
Jones et al., 2000). Most of these laboratory studies have measured in vitro dry matter
digestibility (IVDMD) or gas production. The improvement in IVDMD in some of these
studies has been smaller than expected or even negative for tanniniferous feeds (Jones
et al., 2000). If PEG binds with some component of the feed to produce an insoluble
complex then it would remain in the residue and cause an underestimation of IVDMD.
However, this would not be the case if any complexes formed at rumen pH were
dissociated in the subsequent acid±pepsin stage (Makkar et al., 1995). Estimation of the
PEG in the residue after Stage 1 and release from the residue in Stage 2 would allow an
assessment of such changes. Use of 14C-PEG offers an accurate method for estimating the
PEG bound to the residue after Stage 2, thus allowing a correction to be made to the
IVDMD measurement (CIVDMD).
We examined the effect of a range of PEG rates on IVDMD with leaf material from
two tropical browse species known to differ in tannin content and IVDMD. We also
measured in vitro nitrogen digestibility (IVND) since although PEG may increase IVND,
the presence of PEG in the residue would not cause bias in this estimate. We also
explored the effect of PEG added at Stages 1 and 2 on digestibility during each stage.
2. Materials and methods
2.1. Plant samples
Samples were the terminal ®ve fully expanded leaves from actively growing shoots of
the tropical shrub legumes Calliandra calothyrsus (CPI1 115690) (Calliandra) and
Leucaena leucocephala cv. Cunningham (Leucaena) growing on an alluvial soil at the
CSIRO Lansdown Research Station, 50 km south of Townsville, north Queensland,
Australia (198400 S, 1468500 E). The plant material was frozen and transported from the
®eld in insulated containers and kept frozen in an anaerobic atmosphere prior to freezedrying. After drying the material was ground to pass a 1 mm screen and analysed for nitrogen.
2.2. In vitro digestibility Ð PEG rate
PEG 4000 solutions, prepared to give various rates in the range of 0±1000 mg PEG/g
sample, were spiked with 14C-PEG 4000 (Amersham, UK) in 2 ml water and added to
0.4 g DW (dry weight) of sample in glass centrifuge tubes. To duplicate samples 40 ml
rumen ¯uid/buffer (1:4) was then added. There were 10 levels of PEG for Calliandra and
®ve levels for Leucaena. Tubes were then incubated in an anaerobic chamber at 398C
using a modi®ed Tilley and Terry (1963) in vitro technique (Jones et al., 1998). After
1
Commonwealth Plant Introduction Number (Australia).
B. Palmer, R.J. Jones / Animal Feed Science and Technology 85 (2000) 259±268
261
Table 1
Treatment sequences for digestibility measurements
Treatment No.
Code
Stage 1 (rumen fluid/buffer)
Stage 2 (acid±pepsin)
1
2
3
4
5
w1
p1
w1w2
w1p2
p1w2
Water
PEG
Water
Water
PEG
Water
PEG
Water
Stage 1 (72 h in rumen ¯uid/buffer), tubes were centrifuged for 20 min at 2500g. Two
samples, each of 1 ml were withdrawn from the supernatant and added to 10 ml of
scintillant (OptiPhase `HiSafe' 31; Fisher Chemicals, UK). Tubes were subsequently
counted for 10 min in a scintillation counter (Wallac 1410 Pharmacia, Finland). Residues
in the tubes were washed with water on a vortex mixer, then centrifuged for 10 min, the
supernatant discarded and the process repeated before adding 40 ml of acid pepsin (2 g
1:10,000 pepsin in 1 l 0.1 M HCl). Each tube was then thoroughly mixed on the vortex
mixer and incubated at 398C for 24 h. Tubes were centrifuged, the supernatant subsampled for counting as before, the remaining supernatant discarded and the residues
washed, dried at 808C for 48 h and weighed. Residues were analysed for nitrogen.
2.3. In vitro digestibility Ð timing of PEG addition
In the second experiment, the same plant samples were used and the same digestion
protocol was followed. Two rates of PEG only were used; 0 (Water) and 160 mg PEG/g
sample. Five treatment sequences were compared (Table 1). IVDMD and IVND were
measured after Stage 1 for the ®rst two treatments and after Stage 2 for the remainder.
2.4. Statistical analyses
Data for IVDMD, CIVDMD and IVND were analysed using ANOVA to test for
differences between individual levels of PEG rate and speciesPEG rate interactions
(SPSS, 1995). For the ®rst experiment, curves were also ®tted to treatment means using
an iterative non-linear regression model of the form
Yb0b1(1ÿexp b2X) (SigmaPlot, 1997).
3. Results
3.1. PEG rate
Labelled PEG released during Stage 2 was not signi®cantly different to zero, indicating
that any PEG bound to the residue in Stage 1 was not released in the acid±pepsin. PEG
bound to the residue was subsequently only measured using the 14C-PEG activity in the
supernatant after Stage 1 digestion.
262
B. Palmer, R.J. Jones / Animal Feed Science and Technology 85 (2000) 259±268
Fig. 1. Effect of rate of PEG addition on IVDMD, CIVDMD and IVND for C. calothyrsus leaf.
For Calliandra, increasing rates of PEG increased IVDMD, CIVDMD and IVND
(Fig. 1) with an asymptotic value of 56.4, 65.1 and 88.1%, respectively. These levels were
all achieved at 160 mg PEG/g DM or less. For Leucaena, there was no signi®cant
response to PEG in terms of IVDMD (69.5%). However, the asymptotic responses for
CIVDMD and the IVND were 78.5 and 86.3% at 80 mg PEG/g DM or less (Fig. 2).
Leucaena had signi®cantly higher IVDMD and CIVDMD than Calliandra at all rates of
Fig. 2. Effect of rate of PEG addition on IVDMD, CIVDMD and IVND for L. leucocephala leaf.
B. Palmer, R.J. Jones / Animal Feed Science and Technology 85 (2000) 259±268
263
Fig. 3. Effect of rate of PEG addition on the level of PEG bound in the residue after Stage 1 in vitro digestion for
C. calothyrsus and L. leucocephala leaf.
PEG addition (Figs. 1 and 2). At zero PEG, IVND of Leucaena was higher than for
Calliandra, but above 80 mg PEG/g DM, there were no differences between species.
The relationships between IVDMD, CIVDMD and IVND and PEG are shown in the
following equations:2
Calliandra
IVDMD 53:2 1:40 3:23 0:250 1 ÿ exp ÿ0:100 0:0318 PEG rate; R2 0:96
CIVDMD 51:6 0:30 13:5 0:32 1 ÿ exp ÿ0:0195 0:00110 PEG rate;
R2 0:99
IVND 53:2 1:40 34:9 1:48 1 ÿ exp ÿ0:0227 0:00230 PEG rate; R2 0:99
Leucaena
IVDMD 69:5 0:27 0:0008 0:00060 PEG rate; R2 0:42
CIVDMD 69:8 1:27 8:74 1:520 1 ÿ exp ÿ0:0299 0:01450 PEG rate;
R2 0:94
IVND 74:4 0:90 11:9 1:05 1 ÿ exp ÿ0:0526 0:02040 PEG rate; R2 0:98
The amount of PEG bound by Calliandra was approximately three times that bound by
Leucaena (105 and 33 mg/g, respectively) (Fig. 3). This is in line with the levels to
support maximum IVND for the two species (Figs. 1 and 2).
2
Value in subscript (within parentheses) is standard error of the coef®cient.
264
B. Palmer, R.J. Jones / Animal Feed Science and Technology 85 (2000) 259±268
The relationship between PEG binding and rate of PEG addition to Calliandra and
Leucaena are shown in the following equations:
Calliandra
PEG binding 105:2 2:17 1 ÿ exp ÿ0:0142 0:00110 PEG rate;
R2 0:99
Leucaena
PEG binding 33:2 0:74 1 ÿ exp ÿ0:0280 0:00280 PEG rate;
R2 0:99
Fig. 4. In vitro digestibility of C. calothyrsus and L. leucocephala leaf following Stage 1 and following Stage 2
digestion in the presence and absence of PEG added at Stage 1 or Stage 2 (see Table 1): (a) dry matter
digestibility; (b) corrected dry matter digestibility, and (c) nitrogen digestibility.
B. Palmer, R.J. Jones / Animal Feed Science and Technology 85 (2000) 259±268
265
Fig. 5. Ammonium N levels in the supernatant after Stage 1 digestion in the presence (p1) and absence (w1) of
PEG added at Stage 1 for C. calothyrsus and L. leucocephala leaf.
3.2. Timing of PEG addition
For all treatments, IVDMD and CIVDMD values for Leucaena were higher than for
Calliandra (Fig. 4). With Leucaena, only treatment 5 gave a signi®cantly higher IVDMD,
whereas with Calliandra the addition of PEG at Stage 1 (treatments 2 and 5) resulted in
higher IVDMD; treatment 1 gave a markedly lower value than the other treatments.
Similar results were obtained for CIVDMD except that with Calliandra treatment 4 (PEG
applied at Stage 2) signi®cantly higher values were obtained than for treatment 3 where
no PEG was added (Fig. 4). For Stage 1, PEG increased IVND (p
85 (2000) 259±268
The effect of PEG addition in vitro on dry matter and
nitrogen digestibility of Calliandra calothyrsus and
Leucaena leucocephala leaf
B. Palmer, R.J. Jones*
CSIRO Tropical Agriculture, Davies Laboratory, PMB Post Of®ce, Aitkenvale, Qld 4814, Australia
Received 8 October 1999; received in revised form 24 January 2000; accepted 15 February 2000
Abstract
A modi®ed two stage in vitro digestion method was used to study the effects of rate of PEG addition
(0±1100 mg/g DM) on dry matter (IVDMD) and nitrogen digestibility (IVND) of freeze dried leaf
materialfrom actively growingshootsofCalliandra calothyrsus (Calliandra)and Leucaena leucocephala
cv. Cunningham (Leucaena). For both species, IVDMD and IVND increased asymptotically with PEG
rate; the IVND response being markedly greater for Calliandra. There was a three-fold greater amount
of PEG bound to the residue after Stage 1 (rumen ¯uid/buffer) digestion with Calliandra than with
Leucaena (105 versus 33 mg/g DM). The presence of PEG in the residue, which was not reduced after
Stage 2 (acid±pepsin) digestion, resulted in a higher residue weight and therefore an underestimated
IVDMD. Quanti®cation of the PEG in the residue enabled a corrected IVDMD (CIVDMD) to be estimated. PEG did not bias estimates of nitrogen digestibility in the same way. In a second experiment,
estimates of CIVDMD and IVND were made after Stage 1 and after Stage 2 in the presence and
absence of PEG at 160 mg/g sample. For Leucaena, there were small, but signi®cant, effects of PEG,
whereas with Calliandra there were large responses to PEG and to timing of addition. When measured
after Stage 1, IVND in the absence of PEG was not signi®cantly different to zero (compared with 57%
for Leucaena), whereas with PEG, IVND was 75% (compared with 68% for Leucaena). The low IVND
for Calliandra was associated with low NH4 N levels in the rumen ¯uid/buffer after Stage 1.
In summary, about 160 mg PEG/g sample is appropriate for most studies with tropical tanniniferous
shrub legumes to estimate any deleterious tannin effect. The use of PEG to estimate this effect on
IVDMD is not valid without accounting for the PEG bound to the residue. For estimation of the adverse
effect of tannins on IVND the use of PEG may be more appropriate. # 2000 Published by Elsevier
Science B.V.
Keywords: In vitro digestibility; Tannin containing shrub legumes; PEG binding; DMD; N digestibility
*
Corresponding author. Tel.: 61-7-47538520; fax: 61-7-47538600.
E-mail address: raymond.jones@tag.csiro.au (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 2 5 - 5
260
B. Palmer, R.J. Jones / Animal Feed Science and Technology 85 (2000) 259±268
1. Introduction
The ability of PEG to bind with condensed tannins (CT) has been used in ®eld and
laboratory studies to measure and to reduce the adverse effects of CT in ruminant diets
(Pritchard et al., 1988; Barry, 1989; Silanikove et al., 1994; McSweeney et al., 1999;
Jones et al., 2000). Most of these laboratory studies have measured in vitro dry matter
digestibility (IVDMD) or gas production. The improvement in IVDMD in some of these
studies has been smaller than expected or even negative for tanniniferous feeds (Jones
et al., 2000). If PEG binds with some component of the feed to produce an insoluble
complex then it would remain in the residue and cause an underestimation of IVDMD.
However, this would not be the case if any complexes formed at rumen pH were
dissociated in the subsequent acid±pepsin stage (Makkar et al., 1995). Estimation of the
PEG in the residue after Stage 1 and release from the residue in Stage 2 would allow an
assessment of such changes. Use of 14C-PEG offers an accurate method for estimating the
PEG bound to the residue after Stage 2, thus allowing a correction to be made to the
IVDMD measurement (CIVDMD).
We examined the effect of a range of PEG rates on IVDMD with leaf material from
two tropical browse species known to differ in tannin content and IVDMD. We also
measured in vitro nitrogen digestibility (IVND) since although PEG may increase IVND,
the presence of PEG in the residue would not cause bias in this estimate. We also
explored the effect of PEG added at Stages 1 and 2 on digestibility during each stage.
2. Materials and methods
2.1. Plant samples
Samples were the terminal ®ve fully expanded leaves from actively growing shoots of
the tropical shrub legumes Calliandra calothyrsus (CPI1 115690) (Calliandra) and
Leucaena leucocephala cv. Cunningham (Leucaena) growing on an alluvial soil at the
CSIRO Lansdown Research Station, 50 km south of Townsville, north Queensland,
Australia (198400 S, 1468500 E). The plant material was frozen and transported from the
®eld in insulated containers and kept frozen in an anaerobic atmosphere prior to freezedrying. After drying the material was ground to pass a 1 mm screen and analysed for nitrogen.
2.2. In vitro digestibility Ð PEG rate
PEG 4000 solutions, prepared to give various rates in the range of 0±1000 mg PEG/g
sample, were spiked with 14C-PEG 4000 (Amersham, UK) in 2 ml water and added to
0.4 g DW (dry weight) of sample in glass centrifuge tubes. To duplicate samples 40 ml
rumen ¯uid/buffer (1:4) was then added. There were 10 levels of PEG for Calliandra and
®ve levels for Leucaena. Tubes were then incubated in an anaerobic chamber at 398C
using a modi®ed Tilley and Terry (1963) in vitro technique (Jones et al., 1998). After
1
Commonwealth Plant Introduction Number (Australia).
B. Palmer, R.J. Jones / Animal Feed Science and Technology 85 (2000) 259±268
261
Table 1
Treatment sequences for digestibility measurements
Treatment No.
Code
Stage 1 (rumen fluid/buffer)
Stage 2 (acid±pepsin)
1
2
3
4
5
w1
p1
w1w2
w1p2
p1w2
Water
PEG
Water
Water
PEG
Water
PEG
Water
Stage 1 (72 h in rumen ¯uid/buffer), tubes were centrifuged for 20 min at 2500g. Two
samples, each of 1 ml were withdrawn from the supernatant and added to 10 ml of
scintillant (OptiPhase `HiSafe' 31; Fisher Chemicals, UK). Tubes were subsequently
counted for 10 min in a scintillation counter (Wallac 1410 Pharmacia, Finland). Residues
in the tubes were washed with water on a vortex mixer, then centrifuged for 10 min, the
supernatant discarded and the process repeated before adding 40 ml of acid pepsin (2 g
1:10,000 pepsin in 1 l 0.1 M HCl). Each tube was then thoroughly mixed on the vortex
mixer and incubated at 398C for 24 h. Tubes were centrifuged, the supernatant subsampled for counting as before, the remaining supernatant discarded and the residues
washed, dried at 808C for 48 h and weighed. Residues were analysed for nitrogen.
2.3. In vitro digestibility Ð timing of PEG addition
In the second experiment, the same plant samples were used and the same digestion
protocol was followed. Two rates of PEG only were used; 0 (Water) and 160 mg PEG/g
sample. Five treatment sequences were compared (Table 1). IVDMD and IVND were
measured after Stage 1 for the ®rst two treatments and after Stage 2 for the remainder.
2.4. Statistical analyses
Data for IVDMD, CIVDMD and IVND were analysed using ANOVA to test for
differences between individual levels of PEG rate and speciesPEG rate interactions
(SPSS, 1995). For the ®rst experiment, curves were also ®tted to treatment means using
an iterative non-linear regression model of the form
Yb0b1(1ÿexp b2X) (SigmaPlot, 1997).
3. Results
3.1. PEG rate
Labelled PEG released during Stage 2 was not signi®cantly different to zero, indicating
that any PEG bound to the residue in Stage 1 was not released in the acid±pepsin. PEG
bound to the residue was subsequently only measured using the 14C-PEG activity in the
supernatant after Stage 1 digestion.
262
B. Palmer, R.J. Jones / Animal Feed Science and Technology 85 (2000) 259±268
Fig. 1. Effect of rate of PEG addition on IVDMD, CIVDMD and IVND for C. calothyrsus leaf.
For Calliandra, increasing rates of PEG increased IVDMD, CIVDMD and IVND
(Fig. 1) with an asymptotic value of 56.4, 65.1 and 88.1%, respectively. These levels were
all achieved at 160 mg PEG/g DM or less. For Leucaena, there was no signi®cant
response to PEG in terms of IVDMD (69.5%). However, the asymptotic responses for
CIVDMD and the IVND were 78.5 and 86.3% at 80 mg PEG/g DM or less (Fig. 2).
Leucaena had signi®cantly higher IVDMD and CIVDMD than Calliandra at all rates of
Fig. 2. Effect of rate of PEG addition on IVDMD, CIVDMD and IVND for L. leucocephala leaf.
B. Palmer, R.J. Jones / Animal Feed Science and Technology 85 (2000) 259±268
263
Fig. 3. Effect of rate of PEG addition on the level of PEG bound in the residue after Stage 1 in vitro digestion for
C. calothyrsus and L. leucocephala leaf.
PEG addition (Figs. 1 and 2). At zero PEG, IVND of Leucaena was higher than for
Calliandra, but above 80 mg PEG/g DM, there were no differences between species.
The relationships between IVDMD, CIVDMD and IVND and PEG are shown in the
following equations:2
Calliandra
IVDMD 53:2 1:40 3:23 0:250 1 ÿ exp ÿ0:100 0:0318 PEG rate; R2 0:96
CIVDMD 51:6 0:30 13:5 0:32 1 ÿ exp ÿ0:0195 0:00110 PEG rate;
R2 0:99
IVND 53:2 1:40 34:9 1:48 1 ÿ exp ÿ0:0227 0:00230 PEG rate; R2 0:99
Leucaena
IVDMD 69:5 0:27 0:0008 0:00060 PEG rate; R2 0:42
CIVDMD 69:8 1:27 8:74 1:520 1 ÿ exp ÿ0:0299 0:01450 PEG rate;
R2 0:94
IVND 74:4 0:90 11:9 1:05 1 ÿ exp ÿ0:0526 0:02040 PEG rate; R2 0:98
The amount of PEG bound by Calliandra was approximately three times that bound by
Leucaena (105 and 33 mg/g, respectively) (Fig. 3). This is in line with the levels to
support maximum IVND for the two species (Figs. 1 and 2).
2
Value in subscript (within parentheses) is standard error of the coef®cient.
264
B. Palmer, R.J. Jones / Animal Feed Science and Technology 85 (2000) 259±268
The relationship between PEG binding and rate of PEG addition to Calliandra and
Leucaena are shown in the following equations:
Calliandra
PEG binding 105:2 2:17 1 ÿ exp ÿ0:0142 0:00110 PEG rate;
R2 0:99
Leucaena
PEG binding 33:2 0:74 1 ÿ exp ÿ0:0280 0:00280 PEG rate;
R2 0:99
Fig. 4. In vitro digestibility of C. calothyrsus and L. leucocephala leaf following Stage 1 and following Stage 2
digestion in the presence and absence of PEG added at Stage 1 or Stage 2 (see Table 1): (a) dry matter
digestibility; (b) corrected dry matter digestibility, and (c) nitrogen digestibility.
B. Palmer, R.J. Jones / Animal Feed Science and Technology 85 (2000) 259±268
265
Fig. 5. Ammonium N levels in the supernatant after Stage 1 digestion in the presence (p1) and absence (w1) of
PEG added at Stage 1 for C. calothyrsus and L. leucocephala leaf.
3.2. Timing of PEG addition
For all treatments, IVDMD and CIVDMD values for Leucaena were higher than for
Calliandra (Fig. 4). With Leucaena, only treatment 5 gave a signi®cantly higher IVDMD,
whereas with Calliandra the addition of PEG at Stage 1 (treatments 2 and 5) resulted in
higher IVDMD; treatment 1 gave a markedly lower value than the other treatments.
Similar results were obtained for CIVDMD except that with Calliandra treatment 4 (PEG
applied at Stage 2) signi®cantly higher values were obtained than for treatment 3 where
no PEG was added (Fig. 4). For Stage 1, PEG increased IVND (p