Deinum, B.

Groot, J.C.J.

Lantinga, E.A.

Neuteboom, J.H.

Name not available

Digestibility of cell walls of ageing grass leaves as estimated from in vitro and gas production techniques.

Wageningen Yield 

\ 
Grassland and Society 
Proceedings of the 15th General Meeting of the 
European Grassland Federation, 
June 6-9, 1994 
L. 't Mannetje 
J. Frame 
Editors in chief 
Digestibility of cell walls of ageing grass leaves as estimated from in vitro and 
gas production techniques 
Jeroen C.J. Groot, Hauke Deinum, Egbert A. Lantinga 
and Jan H. Neuteboom 
WJ c . Wageningen Pers Wageningen 1994 

Digestibility of cell walls of ageittg grass leaves as es-
timated from irl vitro attd gas production techniques 
JEROEN C.J. GRooT', BAUKE DEINUM 1 , En BERT A. LANTINGA 2 AND JAN H. NEUTEBOOM 1 
1 Depart;nent (~{Agronomy, Wageningen Agricultural University, Haarweg 333, 6709 
RZ Wa!{eningen, The Netherlands; 2 Department (~l Theoretical Production Ecology, 
Wageningen Agricultural University, Bornsesteeg 65, 6708 PD Wageningen, The 
Netherlands 
Suntmary 
The utilization of roughages is largely dependent on the digestion of cell walls (CW). 
The dynamics of digestion of CW of a leaf of Lolium multU7orum at different stages of 
maturity were quantified. CW digestibility was estimated from in vitro and gas pro-
duction techniques. Ageing of the leaf resulted in a slightly lower rate of in vitro gas 
production from CW fermentation. The rate constant was reduced and the half time in-
creased. Total gas production per grmn CW digested after 120 hours increased with 
age of the leaf. CW digestibility after 4R hours in l'itro was underestimated when cal-
culated from gas production measurements. The changes in gas production parameters 
with increasing CW maturity and underestimation of digestibility from gas production 
were probably caused by the increasing resistance of CW to degradation and by alter-
ations in the contribution of different fermentation processes to CW degradation. 
Keywords: cell walls, digestion kinetics, fermentative gas production, in vitro digest-
ibility, ita I ian ryegrass, Lol i um mult Ulorum. 
Introduction 
Recent changes in intensive grassland management practices toward lower fertilizer 
inputs have caused a shift of interest from increasing production to improving feeding 
quality. Therefore, a better understanding is required of the characteristics of CW in 
degradation processes during rumen digestion, since CW are the least degradable part 
of roughages. In a glasshouse experiment the CW digestion characteristics of plant or-
gans were related to morphological and physiological features of initiation, ageing and 
senescence. 
CW prin1arily consist of structural polysaccharides, which are fermented by rutnen 
microbes, to produce n1icrobial matter, volatile fatty acids and gasses (C02 and CH4). 
The rate and extent of digestion can be estitnated frotn the disappearance of substrate 
0tGESTIBILITY OF CELL WALLS AF AGEING GRASS LEAVES 
or the formation of fermentation end-products in l'itro. In this paper the in vitro diges-
tion n1ethod according to Goering & Van Soest ( 1970) was compared with the cutnula-
tive gas production technique described by Theodorou eta!. (in press). 
Materials and ntethods 
Italian rye grass (Lolium mult(florum) was sown in August 1992, in a glasshouse at 
18/13°C day/night tetnperature. When the ligule of the fourth leaf on the main tiller 
was visible, the plants were cut 2 em above ground level. Plants remained vegetative. 
Leaves from insertion level 7 on the main tiller were harvested when first fully ex-
panded (ligule visible) and at 14, 35 and 49 days thereafter. Samples were oven dried 
(70°C), weighed and ground. Cell wall digestibi Iity ( D4xvitro) was determined using the 
tnethod described by Goering & Van Soest ( 1970). 
Isolated CW (neutral detergent and protease treated) were incubated with rmnen 
fluid for 120 hours and gas production was recorded, according to Theodorou eta!. (in 
press). The resulting cumulative gas production curve was fitted using a sigmoidal 
'switch-on' response curve: 
Y= (1-LY y =-
I (H-LY+(t-L)C 
In this equation, Y1 denotes the yield of gas at timet (hours) after incubation per g 
digested CW after 120 hours ( ml.g DCW- I)~ Y no represents the asymptotic gas produc-
tion (ml.g DCW- 1 ). Lis the lag phase (hours): H is the time after incubation at which 
half of the final amount of gas has been formed (hours) and cis a constant determining 
the sharpness of the switching characteristic. After the gas production measuretnent, 
the proportion of CW digested was determined (0 120 ). From 0 120 and the ratio be-
tween Y 48 andY 12<h the CW digestion after 4X hours was calculated (D4sgas). 
Results 
In Table I the parameters of gas production and estimates of cell wall digestibility are 
presented. Maturation of leaf 7 resulted in a decline in the rate of gas production. The 
value of c decreased with subsequent harvests, and H and L increased. Also Y 120 and 
Y 00 increased. The effects of maturation are shown in Figure I. 
The digestibility of CW after 4R and 120 hours decreased with ageing of the leaf. 
The calculated D4xgas resulted in lower estimates of CW digestibility than D4svitro· This 
discrepancy tended to increase with ageing. 
J. GROOT, 8. DEINUM, E. LANTING/\ AND J. NEUTEBOOM 
Table /. Parameters l~l RllS production .fi·om isolated cell walls l~f'leqf'7 at d~fferent 
dates after appearance l~(the ligule, with standard errors l~f'means (SEM). Significant 
differences (11tkey, a=0.(J5) are indicated u·ith superscripts. 
~ ~ ~ --- - ------~-- ·-- -- -· -- ~ ---
Parameter Days after appearance of the ligule SEM 
-----~---------------- ------~-
() 14 35 49 
--- -
368h 364ah Y 120 (ml/g DCW) 360a 358a 2.72 
Y = (ml/g DCW) 366a 366" 386h 385b 1.73 
H (hours) l9.oa 20.6h 23.4<: 22.9c 0.32 
L (hours) 6.7a 8.6h 10.2<: 9.8c 0.41 
c (-) 1.8oa 1.57h 1.38<: 1.36c 0.07 
0120 (%) 90.9a 88.5h 85.7<: 85.2<: 0.37 
D48vitro (%) 84.9a 80.8h 79.6h 78.1 h 1.24 
D48gas (%) 82.9a 78.4 11 72.8<: 73.2c 0.90 
-------- --------
~ ~ 
...- 400 s 
0 
0 
0> 
::::::::: 
g 300 
"0 
0 
!..... 
0.. 
(/) 
ctS 200 0> 
E 
::J 
0 
100 
0 
0 20 40 60 80 100 120 140 
Time (hours after incubation) 
Figure /. Cumulative gas production l~( isolated cell ~\'ails l~( leq( 7 in a younf? ( D) 
and tnature ( • ) stage ( respectil•ely 0 and 4<) days qfier appearance). Symbols and 
lines represent measured values and.fltted cun·es, respectively. 
0JGESTIBI LITY OF CELL WALLS AF AGEING GRASS LEAVES 
Discussion 
The rate and extent of digestion were reduced with ageing of the leaf. This was prob-
ably caused by changes in CW properties (Fry, 19XR ), which increased the resistance 
of CW to tnicrobial breakdown. The changes in CW characteristics are probably not 
due to changes in chemical composition, hut to an increase in the interpolymeric bond-
ing (Fry, 1988). The rate of decline in digestibility during ageing was low, which is· 
usually observed in such leaves of higher insertion level ( Nordang eta!., I 992). 
Several pathways of microbial fermentation occur in the rumen. Per g DCW, fer-
mentation end-products are produced in different amounts and proportions (Van Hou-
tert, I 993 ). The final gas production g DCW- 1 gives an indication of the contribution 
of the various fermentation pathways to CW degradation. When CW degradability de-
clines, fern1entation shifts to a pathway yielding more gas g DCW- 1 (Beuvink & 
Spoelstra, 1992). This can explain the higher final gas production g DCW- 1 with in-
creasing n1aturity, observed in this experiment. 
Shifts between pathways may occur in the course of the incubation period. Gas pro-
duction from easily degradable substrate fermented in early stages of fermentation 
will be low relative to gas production per g DCW between 4R and 120 hours after incu-
bation. Additionally, when substrate availability decreases, turnover of microbial mat-
ter after lysis might occur, resulting in extra gas production. Consequently, D4sgas 
underestimated CW digestibility. These phenomena are enhanced when CW degrada-
bility declines. Therefore, the difference between D--txvitro and D4xgas increased with 
ageing. 
The cmnulative gas production technique might prove a use full technique for assess-
ing the degradation of roughages and the resulting formation of fermentation products. 
However, the relation between substrate disappearance and production of volatile fatty 
acids, microbial matter and gasses during fermentation has to be established. 
Acl{nowledgen1ents 
We thank Barbara A. Williams from the Department of Animal Nutrition of the 
Wageningen Agricultural University for the use of the gas measurement equipn1ent 
and assistance with the tneasurements. Also the assistance by E.N. van Loo, A. 
Maassen, H. Halm and C. de Jong is kindly acknowledged. 
References 
Beuvink, J .M. W. & S.F. Spoelstra, I YY2. J nteract ions bet ween su hstrate, fermentation end-pro-
ducts, buffering systems and gas production upon fermentation of different carbohydrates by 
mixed rumen microorganisms in l'itro. ;\/'Plied Microhiology and Biotechnology 37: 505-
509. 
Fry, S.C., 1988. The growing plant cell wall: chemical and metabolic analysis. Longman Scien-
t(flc and Technical, HarloH', Essex, 350 p. 
]; GROOT, B. DEINUM, E. LANTING/\ AND J. NEUTEBOOM 
Goering, H.K. & P.J. van Soest. 1970. Forage fiber analysis. Agricultural Handbook 379. 
United States Department f~/Agriculll!re, 20 p. 
Nordang, L., E. A. Lantinga & J.H. Neuteboom, 1992. Development of digestibility of grass leaf' 
blades. Proceedings fi the 14th General Meeting f~j' the European Grassland Federation, 
Lathi, Finland, p. 604-605. 
Theodorou, M.K., B.A. Williams, M.S. Dhanoa, A.B. Mc/\llcn & .J. France. A simple gas pro-
duction method using a pressure transducer to determine the fermentation characteristics of 
ruminant feed. Animal Feed Science and Technology (in press). 
Van Houtert, M.F.J., 1993. The production and metabolism of volatile fatty acids by ruminants 
fed roughages: a review. Animal Feed Science and Technology 43: I R9-225. 


Digestibility of cell walls of ageing grass leaves as estimated from in vitro and gas production techniques.

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