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Journal of the Science of Food and Agriculture
J Sci Food Agric 80:484±490 (2000)
Effects of ageing on the in vitro fermentation of
cell walls and cell contents of entire, fractionated
and composite leaves of Italian ryegrass
Barbara A Williams,1* Arno J Oostdam,1,2† Jeroen CJ Groot,2 Huug Boer1 and
Seerp Tamminga1
1
Wageningen Institute of Animal Sciences (WIAS), Department of Animal Nutrition, Agricultural University, Marijkeweg 40, NL-6709 PG
Wageningen, The Netherlands
2
CT de Wit Graduate School for Production Ecology (PE), Department of Agronomy, Agricultural University, Haarweg 333, NL-6709 RZ
Wageningen, The Netherlands
Abstract: Changes in fermentability of cell walls and cell contents of Italian ryegrass leaves at two
stages of maturity were measured to explain the generally observed decline in herbage quality with
ageing. A herbage fractionation method was developed to separate cell contents and cell walls. Cell
walls were either untreated or protease-treated. Fermentation characteristics of all cell wall and cell
contents fractions, as well as whole and recombined leaves, were measured using cumulative gas and
volatile fatty acid (VFA) production. The gas production pro®les of all substrates contained two
signi®cant phases. After fractionation, addition of the pro®les from separated cell contents and
untreated cell walls resulted in the same pro®le as for the recombined leaf. The strongest reduction in
gas and VFA production due to leaf ageing was observed for cell contents. The increased ratio between
branched and straight chain VFAs suggested that there had been an increase in the protein fermentation. Treatment of cell walls may have removed some easily fermentable cell wall components, as
seen in the small differences in gas and VFA production between whole and recombined leaves. It has
been concluded that the phases of gas production, separable in kinetic fermentability studies of
complex animal feeds, need to be interpreted with caution. The large reduction in fermentability of cell
contents with plant maturity, compared with the cell walls, indicated the importance of determining
the role of cell contents in herbage quality studies, as the cell contents clearly do not remain uniform.
# 2000 Society of Chemical Industry
Keywords: cell walls; cell contents; cumulative gas production; fermentability; plant maturity; protein; ryegrass;
volatile fatty acids
INTRODUCTION
The nutritive value of grass is most often evaluated in
terms of its in vitro organic matter digestibility. This
value is calculated on the assumption that fermentation of the degradable components is complete, which
means that the cell contents, which are soluble, are
always assumed to be 100% degradable. The degradable cell wall fractions, on the other hand, are considered to be the fraction which varies. The actual rate
and extent of fermentation can be estimated from the
gas produced during incubation, as measured using
the cumulative gas production technique.1
The rate of gas production often varies throughout
the incubation period, resulting in consecutive phases
in the gas production pro®les. Mathematical techniques have been developed to separate these
phases.2,3 It has been postulated that phases with high
rates of gas production in the early stages of incubation
are related to an easily fermentable and soluble
fraction. In later stages of fermentation a gradual shift
usually occurs towards the fermentation of insoluble
parts such as plant cell walls.4,5 However, no
information is available concerning the individual
contributions of cell contents and cell walls to gas
production, and the relationship of these relative
quantities to plant composition and degradability.
Separation of the leaf fractions would mean that one
could obtain separate fermentation patterns for the cell
contents and cell walls. These could then be compared
with the two phases of gas production of whole leaf to
determine whether they correspond or whether the
individual fractions behave differently as a result of
being separated. Methods already exist to isolate plant
cell walls. This can be done by a chemical treatment
* Correspondence to: Barbara A Williams, Wageningen Institute of Animal Sciences (WIAS), Department of Animal Nutrition, Agricultural
University, Marijkeweg 40, NL-6709 PG Wageningen, The Netherlands
E-mail: barbara.williams@alg.vv.wau.nl
†
Current address: Cehave nv, PO Box 200, NL-5460 BC Veghel, The Netherlands
(Received 14 June 1999; revised version received 20 September 1999; accepted 4 November 1999)
# 2000 Society of Chemical Industry. J Sci Food Agric 0022±5142/2000/$17.50
484
Fermentation of cell walls and contents of ryegrass
such as the neutral detergent residue method of Van
Soest,6 or by an enzymatic treatment such as pepsin
incubation at low pH.7 Both methods destroy the cell
contents and can alter the cell wall structure and
chemical composition. Therefore it has been necessary
to develop a method of separation which could allow
both cell contents and cell walls to remain intact.
To determine the relationship between crop development and its fermentability, one must also quantify
the effects of environmental factors and plant maturity
on fermentability. In these studies, homogeneous,
evenly aged leaf samples were used in order to avoid
the confounding effects of tillering on leaf age. Therefore leaves from the same position (insertion level) on
the main stem of the grass plants have been used.
The ®rst aim of this work has been to separate the
cell walls and cell contents from leaves of Italian
ryegrass (Lolium multi¯orum Lam) of differing maturity. The second aim has been to determine whether the
assumption that the ®rst phase of gas production
corresponded with the cell contents fraction and the
second with the cell walls was correct. The third and
®nal aim has been to determine whether differences in
fermentability of the components occur owing to the
age of the plant. The cumulative gas production
technique has been used to determine the kinetics of
fermentation. The components have been evaluated as
separated individual components, and as the intact
leaves and the recombined separated cell walls and cell
contents. The recombined components have been
included as a comparison with whole leaves to check
the separation technique.
MATERIALS AND METHODS
Cultivation of Italian ryegrass
Planting and growth conditions of the Italian Ryegrass
(Lolium multi¯orum Lam) cv Multimo (4n) used have
been fully described by Groot et al. 5 Brie¯y, the plants
were harvested after 113 days of growth, when they
had formed eight leaves on the main stem. These
leaves were then dissected from the stem and frozen
separately prior to analysis. Leaves were counted from
top to bottom, so that the last fully expanded leaf
(youngest) has been considered as leaf 1 (L1), and leaf
4 (L4) as a mature leaf.
Separation of leaves into cell contents and cell walls
Following some preliminary trials, the following
method has been developed to separate cell contents
(CC) and cell walls (CW). Fresh leaves were chopped
into 1±2 cm pieces using a paper guillotine, with addition of dry ice, and then ground further in a Knifetec
Sample Mill (Tecator, Sweden). This material was
pressed (HAPA hydraulic press, IKA, Germany;
250 ml) through a double nylon cloth (Nybolt PA
40/30) under a pressure of 100 atm. The residue was
mixed thoroughly with distilled water (8 ml gÿ1 dry
matter) and the process was repeated three times. The
resultant ®ltrate (CC) was cooled immediately with
J Sci Food Agric 80:484±490 (2000)
Table 1. Neutral detergent fibre (NDF), crude protein (CP) and ash contents
(expressed in g kgÿ1 DM) of whole and recombined leaf and cell contents
(CC), cell walls (CW) and protease-treated cell walls (pCW) of leaves L1 and
L4
Fraction
Whole
Recombined
CC
CW
pCW
Leaf
NDF
SE
CP
SE
Ash
SE
1
4
1
4
1
4
1
4
1
4
320
445
298
432
ND
ND
670
814
771
860
0.3
5.0
244
150
246
145
258
226
232
74
120
48
1.1
1.9
106
183
125
198
213
375
18
44
18
44
0.3
1.1
23.5
3.4
14.7
3.6
18.2
0.2
16.3
3.1
7
0.5
6.1
5.0
0.6
0.1
0.3
0.1
dry ice and placed in a ÿ40 °C freezer within 1 h of
collection. The residue (CW) was washed twice with
distilled water (10 ml gÿ1 dry matter). The washed
®ltrates were discarded and the residue was placed in
the freezer within 1 h. The ®ltrate (CC) and residue
(CW) were freeze-dried and ground through a 1 mm
sieve (Retsch mill, Haan, Germany).
The whole leaf substrate was prepared by chopping,
followed by freeze-drying and grinding. Preliminary
experiments had shown that the nitrogen content of
the CW for L1 was about the same as for the total leaf
(see Table 1). To reduce the CW protein content, a
portion of the CW was therefore treated with protease
(Sigma Protease Type XXIII P-4032). This was done
after the water washing of the CW, for 1 h in 60 ml
phosphate buffer containing 0.25 ml protease gÿ1 CW
dry matter. Following protease treatment, the substrate (pCW) was further treated as for CW.
Neutral detergent ®bre (NDF), crude protein, crude
ash and dry matter determinations were carried out for
all substrates. NDF was determined as described by
Goering and Van Soest,8 followed by protease treatment. Crude protein was measured according to ISO
standard 5983, crude ash according to ISO standard
5984, and dry matter according to ISO standard 6496.
Cumulative gas production
The cumulative gas production technique,1 as modi®ed by Williams et al,9 was used to determine the
fermentation characteristics of whole leaves, CW,
pCW, CC and recombined CW ‡ CC for both L1
and L4. Four replicates from each of the two blocks
were fermented in 50 ml serum bottles containing 0.2 g
DM of each substrate, 41 ml of medium B10 and 2.5 ml
of rumen ¯uid inoculum. The rumen ¯uid inoculum,
obtained from three hay-fed sheep, was prepared as
described by Williams et al. 9 Thirty gas readings
(measuring pressure and volume) were taken over the
120 h incubation period.
After 120 h of incubation the amount of unfermented dry matter was determined by washing the residue
®ve times with hot water (10±15 ml) in a pre-weighed
sintered glass crucible (Pyrex, #2), after which the
485
BA Williams et al
crucibles were ashed at 550 °C for the determination of
organic matter degradation. Neutral detergent residue
(NDR) was determined by treatment of the dry matter
residue with neutral detergent.8 Volatile fatty acid
(VFA) production in the medium was measured using
GLC (Packard 419 glass column (CE Instruments,
Milan, Italy), ®lled with Chromosorb 101, carrier gas
N2 saturated with methanoic acid, at 190 °C, with iso
caproic acid as an internal standard). Acetic, propionic, isobutyric, butyric, valeric and isovaleric acids
were measured. The VFAs are reported in units of
acetic acid equivalents (mg AAE gÿ1 OM). The unit is
calculated by multiplying the amount of acid,in mg, by
the conversion factor (propionic, 1.21; isobutyric and
butyric, 1.36; isovaleric and valeric, 1.46) to give a
result in mg AAE.11 This unit allows an overview of
the energy and/or carbon content of individual VFAs
in relation to each other.
Statistical analyses
Gas production pro®les were ®tted iteratively to the
multiphasic logistic curve2
Gˆ
n
X
iˆ1
1‡
i
ÿ
e i …1ÿi †
where ai is the asymptotic gas production (ml gÿ1
OM), bi is the rate constant (% hÿ1), li is the time axis
intercept of the tangent to the in¯ection point (h) and i
is the number of phases.
The number of phases was determined according to
the method described by Groot et al. 3 The NLREG
package12 was used for the non-linear regression
analysis. The models with a different number of
phases were compared by t-test and F-test analysis.
The signi®cance of individual model parameters was
assessed by testing their t-value (parameter value/
standard error) against tDF (p < 0.05; DF, degrees of
freedom). The signi®cance (p < 0.05) of improvement
in the residual sum of squares (RSS) when the number
of phases in the model was increased from i to i ‡ 1 was
determined by testing13
f ˆ
…RSSi‡1 ÿ RSSi †=…DFi‡1 ÿ DFi †
…DFi‡1 ÿ DFi †
vs F
RSSi =DFi
…DFi †
where DFi is the number of degrees of freedom when i
phases are included in the model. Analyses of variance
were performed using the program SAS,14 and the
following model was used for the statistical analysis of
treatment effects:
Yij ˆ ‡ Li ‡ Bi ‡ Li Bj ‡ eij
where Yij denotes the cumulative gas production, DM
loss, OM loss or VFA production, mis the mean, Li is
the age of the leaf, Bi denotes the block effect, Li * Bj
denotes the interaction between age and block and eij is
the error term.
486
RESULTS
Separation of CW and CC and chemical composition
Table 1 shows the results of NDF, crude protein and
ash analysis for all the substrates used in the gas
production method. NDF was not analysed for the CC
fraction, ®rstly owing to the limited amount of material
available, and secondly because preliminary work had
shown that the NDF content was less than 5 g kgÿ1
OM.
DM weights of the original material and of the
residues and ®ltrates following the separation process
showed that there had been some losses as a result of
the process. These have amounted to 10.5 and 10.9%
of the original material for L1 and L4 respectively
(data not shown). It was assumed that the losses came
equally from both fractions. For L1 and L4 the dry
matter content was 43.8 and 52.9% CW and 56.2 and
47.1% CC respectively.
For all the substrates except CC, the NDF content
was shown to increase with increasing leaf number.
Crude protein was greatly reduced with increasing leaf
number for all the substrates except CC, which had
only a slight decrease. The ash content of all substrates
increased to varying extents with leaf number, though
the increase has been least for CW and pCW. NDF
analysis showed that NDF was the largest component
of the CW fractions. The protease treatment reduced
the crude protein content by almost half for both
leaves, which led to a relative increase in the NDF
content of pCW.
The analysis for whole and recombined substrates
showed that the differences between them were quite
small, though the separation/recombination process
did result in a slightly higher ash and lower NDF
content for both leaves.
Gas production and organic matter digestibility
The OM digestibility (OMD), measured total gas
production (G, ml) and yield of gas (Y, ml gÿ1
disappeared) are shown in Table 2.
OMD, G and Y had a similar pattern to the VFA
yields for all the substrates. Whole, recombined and
Table 2. Organic matter degradability (OMD, %), measured
cumulative gas production (G ml gÿ1 OM) and gas yield (Y, ml gÿ1
degraded OM) of whole and recombined leaf and cell contents
(CC), cell walls (CW) and protease-treated cell walls (pCW) of
leaves L1 and L4
Fraction
Whole
Recombined
CC
CW
pCW
Leaf
OMD
G
SE
Y
SE
1
4
1
4
1
4
1
4
1
4
95.2
91.9
95.0
91.6
100
100
92.2
88.8
82.8
82.6
313
240
270
194
235
81
301
329
262
288
12.5
19.4
15.4
26.5
14.8
12.4
15.6
12.2
11.7
11.0
329
262
284
212
235
81
325
370
316
348
13.0
23.8
16.1
28.9
14.8
12.4
17.0
13.7
12.0
13.3
J Sci Food Agric 80:484±490 (2000)
Fermentation of cell walls and contents of ryegrass
Phase 1
Fraction
Whole
Recombined
Table 3. Parameters of gas production of
whole and recombined leaf and cell
contents (CC), cell walls (CW) and
protease-treated cell walls (pCW) of leaves
L1 and L4; a (ml gÿ1 OM), b (h) and
l (% hÿ1) are the fitted parameters of the
diphasic logistic model
CC
CW
pCW
Phase 2
Leaf
a1
SE
b1
SE
l1
SE
a2
1
4
1
4
1
4
1
4
1
4
181
115
152
97
138
53
165
187
120
163
10.2
19.6
9.3
21.1
6.3
13.9
11.5
12.7
22.6
16.4
33.3
24.0
33.2
27.1
23.1
25.8
43.0
43.5
26.1
24.3
1.65
3.68
1.29
2.89
1.91
2.72
2.78
1.86
4.68
3.90
9.4
14.3
10.0
14.3
10.4
10.8
11.2
12.0
17.9
22.7
0.36
1.33
0.42
1.25
0.38
0.50
0.49
0.76
1.74
1.80
124
118
112
92
95
27
125
131
132
117
CC fractions had reduced G and Y for higher leaf
number and a reduced OMD in the case of the whole
and recombined leaf. Cell wall fractions, on the other
hand, had a slight tendency to an increase in all the gas
parameters with increasing leaf number. The
measured yield of gas for the whole leaf was slightly
higher than for the recombined material, which was in
agreement with the total VFA production values.
The OMD of leaf CC was assumed to be 100%. It
should be remembered, however, that this is really a
matter of being 100% soluble rather than an indication
of their fermentability. However, a difference in
fermentability of the soluble components was strongly
suggested by the fact that there were very signi®cant
differences not only for the gas parameters of different
leaf numbers but also for the VFA yield.
SE
b2
SE
l2
SE
8.7 7.4 0.25 27.3 1.85
10.1 7.8 0.46 36.6 2.87
5.4 6.8 0.21 30.7 1.20
10.0 7.9 0.33 35.5 2.79
11.9 5.9 0.27 35.8 1.72
11.1 14.4 3.82 25.0 4.41
4.9 9.1 0.48 30.0 1.54
2.7 10.1 0.51 29.9 1.67
20.4 8.1 1.12 42.0 9.56
15.0 8.4 0.51 45.8 3.17
The gas production pro®les measured for CC and
CW were added together in the proportions of DM
found during the separation process, and then compared with the recombined leaf. Summation of both
®rst phases of CC and CW corresponded well to the
®rst phase of recombined L1, and the same was true
for the second phase. (Fig 1A). The total gas of phases
1 and 2 of the summed pro®les was 150 and
108 ml gÿ1 OM respectively for L1, which was about
the same as for the `real' recombined leaves (Table 3).
For L4, however, summation of CC and CW overestimated the gas production compared with the
recombined leaves (Fig 1B).
Gas production kinetics
The gas production data were ®tted to a monophasic
curve, and the deviations of data from the ®tted curve3
made it clear that there were two phases present for all
the substrates, except for pCW which had three (not
reported here). The signi®cance of adding the second
phase was con®rmed by the F-test analysis of the RSS
and a t-test of the parameters. Addition of a third
phase signi®cantly (p < 0.05) reduced the RSS of every
fraction, except L4 CC, but few of the extra parameters were signi®cant. Hence it is the results of the
diphasic models which are reported here (Table 3).
The distribution of the gas production between the
®rst and second phases was the same for both whole
and recombined leaves. The reduced gas production
of L4 compared with L1 was most pronounced in the
®rst phase. About 58 and 50% of the total gas was
produced in the ®rst phase for L1 and L4 respectively.
There were no other differences in the gas production
kinetics (b and l) between whole and recombined
leaves.
The gas volume estimated for both phases was signi®cantly (p < 0.05) different for the different fractions. CC had the lowest gas volume for both phases
and the steepest reduction between L1 and L4.
Limited differences were observed for the kinetic
parameters (b and l) according to substrate and leaf
number.
J Sci Food Agric 80:484±490 (2000)
Figure 1. Gas production profiles for recombined leaf (&), for cell contents
(CC, *) and cell wall (CW, !) fractions, according to their proportion in
DM, and summation of CC and CW (CW ‡ CC, &) for L1 (A) and L4 (B).
487
BA Williams et al
Table 4. Means and standard deviations (in parentheses) of VFAs present at end of fermentation in AAE gÿ1 OM, with total in AAE gÿ1 OM (A) and mmol gÿ1 OM
(B), and ratio of branched chain (isobutyric, isovaleric and valeric) to non-branched chain (acetic, propionic and butyric) fatty acids (BCR)
Fraction
Whole
Recombined
CC
CW
pCW
Leaf
1
4
1
4
1
4
1
4
1
4
Acetic
381
372
359
375
319
320
370
381
360
357
(9)
(25)
(31)
(20)
(31)
(37)
(26)
(17)
(19)
(10)
Propionic
243 (3)
213 (12)
245 (4)
218 (23)
314 (13)
204 (14)
227 (35)
227 (10)
177 (40)
272 (7)
Isobutyric
29
19
25
20
18
14
29
18
23
21
(1)
(6)
(4)
(3)
(4)
(6)
(3)
(2)
(1)
(2)
VFA production
VFA production at the end of fermentation is shown
for each substrate in Table 4 (mg AAE gÿ1 OM). This
unit was used as it allows one to place all the VFAs on
equal footing in terms of carbon and energy content.11
The ratio of the sum of the branched chain acids plus
valeric (in AAE) to the sum of acetic, propionic and
butyric (BCR, branched chain ratio) is also shown, as
is the total VFAin mmol Iÿ1.
For all the substrates except pCW there was a
decrease in the total VFA produced from L4 compared
with L1. This decrease was quite small in the case of
the cell wall fractions, but became more pronounced
for the substrates with a greater proportion of cell
contents (whole and recombined substrates). The
decrease was most marked for CC alone. For CC this
decrease was most marked for propionic and butyric
acids, while acetic acid concentrations remained unchanged. The change in the ratio of branched chain to
non-branched chain VFAs was most marked for CC
and CW and was most likely related to the protein
content, as can be seen in Fig 2.
The CW fractions themselves have been very similar
in terms of their VFA production, though the slight
differences did put them into an order of CW > pCW
for leaf 1 and pCW > CW for leaf 4. The CC had a
completely different VFA pattern to the cell wall
fractions, with a greater proportion of propionic acid
for the younger leaf.
The fact that the total VFA produced was slightly
higher for the whole leaf compared with the recombined material suggests that the recombination process
was not perfect and that some losses occurred,
particularly from the younger material. This difference
was not signi®cant, but was most pronounced for
acetic acid.
Butyric
108 (2)
79 (6)
105 (7)
78 (8)
100 (6)
33 (2)
103 (4)
86 (5)
76 (6)
66 (6)
Isovaleric
73
52
66
43
55
31
73
42
53
53
(3)
(10)
(6)
(6)
(15)
(6)
(4)
(4)
(3)
(5)
Valeric
61
55
59
51
65
22
57
37
46
44
(3)
(6)
(4)
(5)
(8)
(7)
(8)
(4)
(3)
(5)
Total A
Total B
BCR
895 (5)
790 (56)
860 (55)
785 (55)
871 (56)
623 (64)
858 (14)
791 (40)
734 (55)
812 (30)
11.1 (0.1)
10.1 (0.7)
10.7 (0.7)
10.1 (0.7)
10.6 (0.7)
8.4 (0.9)
10.7 (0.12)
10.3 (0.5)
9.5 (0.7)
10.4 (0.3)
0.2228
0.1901
0.2118
0.1698
0.1883
0.1203
0.2273
0.1399
0.1992
0.1698
these substrates (both separated and whole), to determine the extent to which the two phases corresponded
to the separated cell walls and cell contents. The last
aspect was to gain information concerning changes in
fermentability of leaves with maturity, in terms of both
the separated components and the whole material.
Many methods exist to separate cell walls (eg the
neutral detergent method of Van Soest6) from forage,
but until recently there seemed to be little interest in
separating cell walls and cell contents from each other,
resulting in two fractions to work with. This may be
because the in vitro techniques currently in common
use rely on changes in particle size and so do not lend
themselves to an examination of cell contents, which
are mostly soluble. However, with the increasing use of
cumulative gas production methods it was a logical
next step to consider the CC fraction of the plant in
conjunction with the CW as well, particularly since the
development of multiphasic models of analysis.2,3
The method described here gave a good separation
of grass leaves into their cell walls and cell contents,
DISCUSSION
There were three main aspects developed in this work.
The ®rst was a technique to separate cell walls and cell
contents of fresh grass so that they could be fermented
separately, thus allowing us to examine the contribution of each to the nutrition of the ruminant. The
second aspect was the use of a multiphasic model to ®t
488
Figure 2. Relation between substrate crude protein concentration (g kgÿ1)
and Br-VFA (including valeric acid)/NBr-VFA ratio (ratio calculated using
AAE gÿ1 OM).
J Sci Food Agric 80:484±490 (2000)
Fermentation of cell walls and contents of ryegrass
although there were some losses (about 10%). This
was most likely due to leaf material sticking to the
nylon cloth, and could, if deemed necessary, be solved
in the future by more thorough soaking of the cloth,
washing with water only, and including the wash water
with the CC substrate. The treatment with protease
gave an indication of how much protein was associated
with the cell walls, which also became clear later from
the fermentation patterns.
Chemical analysis and incubation with rumen ¯uid
showed signi®cant differences between the separated
components. The ratio of the sum of the branched
chain acids plus valeric to the sum of acetic, propionic
and butyric acids (AAE) was closely related to the CP
concentration (Fig 2), except for CC of L4 (see
below). This is in agreement with the results of EI
Shazly,15 who found higher concentrations of the
branched chain acids in vivo for higher-protein rations.
For L1 the ®rst phases of CC and CW added
together corresponded very well with the ®rst phase of
the recombined leaf, which was also true for the
second phase. The presence of two phases for CC
seems in con¯ict with the work of Cone,4 who
suggested that during the fermentation of complex
substrates the ®rst phase could be strictly related to the
CC fraction of the substrate. These results, on the
other hand, con®rmed the curve subtraction technique
described by Stefanon et al 16 in their work with watersoluble and insoluble components of three legume
species, and showed that it was also valid for ryegrass
leaves. However, a more complex picture emerged in
the case of L4. For the L4 recombined leaf, the
proportion of CW may have been less than in the
original leaf.
The differences in composition and degradability
between grass leaves were considered to be a result of
leaf ageing. L4 had appeared approximately 42 days
before L1. Apart from the difference in maturity, laterformed leaves on the main stem (higher insertion level)
usually have a higher NDF concentration and lower
CWD and OMD when the leaves ®rst appear.17
However, for this work it was assumed that any
possible reduction in the effect of maturity caused by
the difference in insertion level was negligible. This
was supported by the higher NDF and ash concentrations and the lower CP content and OMD observed
for L4, which is in agreement with the work done by
others on the effects of maturity on leaf characteristics.18±21
It seems that the decrease in the production of gas
from the leaves (whole and recombined) with increasing age could mainly be attributed to reduced
fermentability of the CC fraction, as the different cell
wall components remained remarkably similar.
Although CP concentration was reduced only slightly
in CC with leaf ageing, the ratio of acetic/propionic/
butyric to other acids was higher. This suggests that,
with maturity of the leaf, protein fermentability
declines in CC. Gutek et al 22 showed that plant
maturity in legumes led to an increase in polyphenolic
J Sci Food Agric 80:484±490 (2000)
compounds, which bound some of the protein present
and therefore made it unavailable for fermentation.
The form of the N which is present may also change
with leaf ageing.23
CONCLUSIONS
Grass leaves can be divided into their cell walls and cell
contents and can be fermented separately. Such
separation is appropriate for those situations where
one is interested not only in the plant cell walls but also
in the cell contents, especially for their chemical
components or fermentative characteristics. Indeed,
until now it has been assumed that the cell contents of
plants are totally digestible and make a uniform
addition to the nutritive value of forages, unlike the
cell wall components which have been recognised as
being very variable according to the species, age and
part of the plant. However, cell contents comprised
between 47 and 56% of the dry weight of the grass
leaves, and this work shows that they make a signi®cant contribution to the fermentation of the whole
leaf, both in terms of gas and VFA production. It has
also been shown that this contribution varies with the
age of the leaves.
Differences in CC and CW of young and old leaf
were readily apparent when fermentation characteristics were measured. For ryegrass it would seem that
CC is actually the more variable component of the
leaves as ageing occurs. This was seen not only from
the much larger decrease in fermentability, as
measured by gas and VFA production for the two
maturities of leaf, but also from the change in VFA
pattern and a signi®cant increase in ash. Just because a
component is soluble, it does not automatically mean
that it is readily utilised by rumen micro-organisms
and can therefore contribute to the energy input of
herbivores. Great caution needs to be taken when
interpreting the fermentation characteristics of protein-rich substrates of different maturity (eg legumes).
Biologically speaking, there are undoubtedly more
underlying phases in cumulative gas production
curves24 which relate to microbial activity and most
probably to their use of particular chemical compounds as substrates. It seems to be possible to detect
mathematically the moment when a visible change
occurs between their use of rapidly fermentable
components to their use of more slowly fermentable
components as substrate, though both of these
elements may be present in both cell walls and cell
contents. However, it must be remembered that there
is not only a question of whether the changeover can
be detected, but, more importantly, whether the
phases, which are detected mathematically, have a
readily apparent biological meaning.
ACKNOWLEDGEMENTS
The authors would like to thank Andre Maassen and
Marianne van 't End for their help with the gas
489
BA Williams et al
readings. Dick Bongers of the Department of Human
and Animal Physiology helped with the VFA analysis.
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