Directory UMM :Data Elmu:jurnal:A:Animal Feed Science and Technology:Vol82.Issue1-2.Nov1999:
Animal Feed Science and Technology
82 (1999) 51±61
Ensiling of whole crop wheat with
cellulase±hemicellulase based enzymes
3. Comparing effects of urea or enzyme treatment
on forage composition and stability
T. Adogla-Bessaa,1, E. Owena, A.T. Adesoganb,*
a
Department of Agriculture, The University of Reading, Earley Gate,
P.O. Box 236, Reading RG6 6AT, UK
b
Welsh Institute of Rural Studies, University of Wales, Aberystwyth, Llanbadarn Campus,
Aberystwyth SY23 4AL, Wales, UK
Received 21 October 1998; received in revised form 25 June 1999; accepted 20 July 1999
Abstract
This work compared the effect of ensiling technique and application of urea or a cellulase±
hemicellulase enzyme mixture (FSO2) on the conservation of whole crop wheat. Awheat crop harvested
at 600 g dry matter (DM) kgÿ1 was conserved with or without the application of urea (40 g kgÿ1 DM)
and four levels of FSO2 (0, 1750, 3500 and 15 503 ml tÿ1 DM). Each forage (5 kg DM) was conserved
in polythene-bag silos that were evacuated and sealed immediately, sealed immediately but not
evacuated or evacuated and sealed after 24 h. After at least 42 days of conservation, the silos were
opened and analysed for chemical composition, rumen fluid-pepsin in vitro digestibility and aerobic
stability. In non-urea-treated forages, increasing enzyme application rate did not affect in vitro
digestibility, but increased water soluble carbohydrate and lactic acid contents, and reduced pH, neutral
detergent fibre, acid detergent fibre and cellulose contents. Compared to the FSO2 only treatments, urea
treatment increased pH and N content and reduced ensiling DM loss, neutral detergent fibre, acid
detergent fibre, acid detergent lignin and cellulose contents. Application of FSO2 and urea (FSO2 U)
produced forages with higher in vitro digestibility and lower cell wall contents than in FSO2 only
forages. NDF and ADF contents were also 5±10% lower in FSO2 U forages than in those conserved
with only urea. Immediate evacuation of silos did not enhance fermentation quality. Delaying silo
sealing by 24 h increased lactic acid content and aerobic stability relative to either of the immediate seal
treatments. Urea treatment alone and the high enzyme level alone also enhanced aerobic stability.
However, increasing the enzyme application rate in the FSO2 U treatments did not enhance
*
Corresponding author. Tel.: 44-1970-624471; fax: 44-1970-611264
E-mail address: ata@aber.ac.uk (A.T. Adesogan)
1
Present address: University of Botswana, Botswana College of Agriculture, Bag 0027, Gaborone, Botswana.
0377-8401/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 9 9 ) 0 0 1 0 0 - 5
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T. Adogla-Bessa et al. / Animal Feed Science and Technology 82 (1999) 51±61
stability. These findings indicate that in whole crop wheat, the medium (3500 ml tÿ1 DM) and high
(15 503 ml tÿ1 DM) levels of FSO2 application are as effective as urea in degrading cell walls and
maintaining aerobic stability. However, nutritive value is optimised when whole crop wheat is
conserved using a mixture of urea and the low level (1750 ml tÿ1 DM) of FSO2. # 1999 Elsevier
Science B.V. All rights reserved.
Keywords: Whole crop wheat; Chemical composition; In vitro digestibility; Aerobic stability
1. Introduction
Whole crop wheat (WCW) can be harvested and successfully conserved over a wide
range of crop maturities (350±700 g DM kgÿ1; Adogla-Bessa and Owen, 1995) but the
optimum dry matter (DM) yield is attained at the medium-dough growth stage (550±600 g
DM kgÿ1) (Corral et al., 1977). However, at such advanced growth stages, water soluble
carbohydrate content (WSC) is low and therefore fermentation capacity is reduced. Urea
application has been successfully used to overcome this problem as the resulting alkaline
preservation obviates the need for a high WSC content in the crop. However, several
studies have shown disappointing production results when dairy cows were fed ureatreated WCW harvested at optimal DM yield (Leaver and Hill, 1992). This problem is
thought to relate largely to the lignified pericarp in mature WCW, which prevents digestion
of the grain. Although cellulolytic enzymes have been successfully used to hydrolyse
forage polysaccharides in grass silage (Huhtanen et al., 1985) and whole crop sorghum
silage (Laytimi et al., 1988), little is known about their potential for conserving and
enhancing the nutritive value of WCW. Therefore, this work aimed to determine how
cellulolytic enzymes compare with urea for conserving mature WCW. To this end, the
study evaluated the effect of urea or enzyme treatment on the chemical composition, in
vitro digestibility and aerobic stability of WCW harvested at optimum DM yield. Since
WCW is particularly prone to aerobic deterioration, this work also investigated the effect
of different ensiling techniques on the conservation of WCW. The study is particularly
relevant to the current UK situation where farmers are looking for alternatives to urea
treatment for conserving WCW harvested at optimal DM yield. This is because of the
inefficient utilisation of such forages due to losses of undigested cereal grains in the faeces.
This study follows on from previous work (Adogla-Bessa and Owen, 1995) which
showed that specific cellulase±hemicellulase enzymes enhanced the fermentation of
immature (< 500 g kgÿ1 DM) WCW but did not enhance digestibility or reduce fibre
content. A different enzyme mixture with a higher hemicellulase activity (FSO2) was
investigated in this study.
2. Materials and methods
2.1. Forage preparation and additive treatments
Whole crop winter wheat (cv. `Brock') was cut and harvested with a New Holland 717
metered-chop, forage harvester, at the medium-dough stage (600 g DM kgÿ1). The crop
T. Adogla-Bessa et al. / Animal Feed Science and Technology 82 (1999) 51±61
53
was precision chopped and conserved with an enzyme additive (FSO2, a cellulase±
hemicellulase experimental enzyme mixture from Finnish Sugar, Helsinki, Finland)
applied at rates of 0, 1750 ml tÿ1 DM (low), 3500 ml tÿ1 DM (medium) and 15 503
ml tÿ1 DM (high). Additional treatments included the application of urea (40 g kgÿ1 DM)
alone and the application of a mixture of urea and each of the enzyme levels (FSO2 U).
The enzyme was obtained in liquid form, diluted to a standard volume and applied using
a high velocity pressure sprayer (Kremlin pneumatic sprayer, model J4, Kremlin Spray
Painting Equipment Ltd. Slough, UK). Enzyme activities (IU mlÿ1) for FSO2 were as
follows: cellulase 2.08; carboxymethyl cellulase 54.00; cellobiase 3.24; xylanase 496.9;
(Jacobs, 1989).
Each forage (5 kg) was conserved in a polythene bag silo using the procedure outlined
by Adogla-Bessa and Owen (1995). The silos were evacuated as completely as possible
with a vacuum pump (Edwards Hi-Vac vacuum pump, Crawley, UK) and sealed
immediately, not evacuated and sealed immediately, or evacuated and sealed after 24 h.
2.2. Forage stability measurements
The forages were ensiled for a minimum of 42 days and ensiling DM loss determined
as the weight difference between silo DM content at sealing and opening. Silos were
exposed for 55 days in a controlled environment room (15±178C). Exposure DM loss was
determined as the weight difference between silo DM content at opening and at the end of
the exposure period. Silo temperatures were monitored daily during the exposure period,
as an index of bacterial activity and aerobic deterioration. Thermocouple wires (Ni±Al) were
inserted into the geometric centre of each silo and left in situ. The wires were connected to a
central junction and recording box (Wheatstone Bridge, Comark 1621, Comark Electronics,
Littlehampton, UK) which enabled temperature to be read without disturbing the silo.
2.3. Chemical analyses
Oven DM was determined by drying 400±500 g of fresh or thawed samples at 55±658C
in a forced draught oven for 48 h. A corrected forage DM was determined by the method
of McDonald and Dewar (1960). A glass electrode (WPA pH meter, WPA, Linton, UK)
was used to determine pH on fresh samples. Total nitrogen (N) was determined by complete combustion of freeze-dried samples in a Leco nitrogen analyser (Leco FP-228, Leco
Corporation, St. Joseph, Michigan, USA). Acid detergent fibre (ADF) and neutral
detergent fibre (NDF) were determined by the method of Van Soest et al. (1991); 2.0 ml
of 2% amylase solution was added to aid NDF filtration. WSCs were determined by
measuring the reducing activity of the sugars (Smith and Groteluschen, 1966) in ovendried samples with a Chemlab continuous-flow colorimeter (Chemlab instruments,
Hornchurch, UK). Volatile fatty acids and lactic acid were assessed by gas
chromatography (Wilson and Wilkins, 1978). In vitro digestible organic matter content
(DOMD) was determined by a modification of the 2-stage method of Tilley and Terry
(1963); 10 mg of nitrogen as ammonium sulphate was added at the start of the first stage,
and microbial activity at the end of the first stage was terminated by filtering and adding
acidified pepsin solution to the sample.
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T. Adogla-Bessa et al. / Animal Feed Science and Technology 82 (1999) 51±61
2.4. Experimental design and statistical analysis
The design was a 2 4 3 factorial arrangement with 2 replicates per treatment. Data
were analysed using the Genstat Statistical Package (Lawes Agricultural Trust, 1988) and
differences between means were tested using the students t-test. F ratios were inspected
and where the main effects were positive and much larger than the interaction effect, they
were also discussed (Mead and Curnow, 1983).
3. Results
3.1. Effect of additive type
Table 1 shows the effect of additive type on forage chemical composition and DM loss.
Silo DM losses during ensiling and exposure were lower (P < 0.01) in forages treated
FSO2 U than in FSO2-treated forages (Table 1). The FSO2 U treatment also gave
higher (P < 0.01) in vitro DOMD and total N contents, and lower (P < 0.05) ADF,
cellulose, ADL and NDF (P < 0.05) contents than the FSO2 treatment. FSO2 U
application also resulted in higher (P < 0.01) pH and acetic acid levels and lower
(P < 0.01) lactic and butyric acid levels. Aerobic spoilage was inhibited for up to 30 days
Table 1
Main effect of enzyme (FSO2) and enzyme urea (FSO2 U) treatment on silo dry matter loss, chemical
composition and fermentation products in WCW forages
SEDb
Additive
pH
In vitro organic matter digestibility
(g kgÿ1 DM)
Silo dry matter loss (g kgÿ1 DM)
Ensiling
Exposure
Composition of dry matter (g kgÿ1 DM)
Total nitrogen
Neutral detergent fibre
Acid detergent fibre
Cellulose
Acid detergent lignin
Fermentation products
Lactic acid (g kgÿ1 DM)
Acetic acid (g kgÿ1 DM)
Butyric acid (g kgÿ1 DM)
Ammonia nitrogen (g kgÿ1 N)
a
b
FSO2a
FSO2 U
4.7
530
8.9
620
11
160
3
126
14.8
492
301
240
52
21.2
441
274
220
49
0.19
13.5
10.9
9
1.8
7.6
5.4
1.9
67
2.4
10.5
0.1
379
0.44
0.34
0.07
10
0.02
11.0
0.2
4.1
An experimental cellulase±hemicellulase enzyme mixture from Finnish Sugar Company, Helsinki, Finland.
Number of observations per mean was 24.
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T. Adogla-Bessa et al. / Animal Feed Science and Technology 82 (1999) 51±61
by FSO2 U application except at the low enzyme level (Fig. 1). Whereas deterioration
was apparent within 10 days in the FSO2-treated forages.
Compared to the FSO2-treated forages, urea treatment increased pH and N content and
reduced ensiling DM loss, NDF, ADF, ADL and cellulose contents (Table 2).
3.2. Effect of enzyme application rate and interaction with additive type
Table 2 shows the effect of enzyme concentration on chemical composition. Increasing
the rate of enzyme application did not affect in vitro DOMD or exposure DM losses. In
FSO2-treated forages, increasing enzyme concentration increased WSC content whilst
Table 2
Effect of enzyme (FSO2a) application rate on chemical composition, in vitro digestibility and fermentation
products in WCW forages conserved with enzymes or with an enzyme±urea mixture (FSO2 U)
SEDb
Enzyme application rate (ml tÿ1 DM)
0
(untreated)
1750
(low)
3500
(medium)
15 503
(high)
153
137
143
138
4.7
8.0
570
4.4
8.1
570
4.3
7.6
580
6.6
8.2
580
0.63
0.42
16.0
FSO2
FSO2 U
FSO2
FSO2 U
11.9
2.8
4.9
8.8
11.4
3.4
4.8
8.6
11.1
3.2
4.7
8.9
9.4
4.1
4.4
8.8
0.31
Total nitrogen
(g kgÿ1 DM)
Neutral detergent fibre
(g kgÿ1 DM)
Acid detergent fibre
(g kgÿ1 DM)
Cellulose
(g kgÿ1 DM)
FSO2
FSO2 U
FSO2
FSO2 U
FSO2
FSO2 U
FSO2
FSO2 U
14.5
21.2
526
448
333
276
250
22
14.3
21.4
526
461
320
262
250
21
14.7
21.3
485
418
288
256
240
20
15.6
20.7
432
469
263
301
200
23
0.39
Water soluble carbohydrates
(g kgÿ1 DM)
Butyric acid
(g kgÿ1 DM)
FSO2
FSO2U
FSO2
FSO2 U
64
77
2.5
0.1
69
82
2.0
0.1
89
82
1.7
0.1
118
76
1.9
0.1
FSO2
FSO2 U
74
87
Main effectsc
Silo dry matter loss during
exposure (g kgÿ1 DM)
Lactic acid
Acetic acid
Digestible organic matter
(g kgÿ1 DM)
Interaction effectsb
Ensiling DM loss (g kgÿ1 DM)
pH
Ammonia-nitrogen (g kgÿ1 N)
a
79
446
57
487
57
498
5.8
0.03
27.1
21.8
17.0
5.3
0.14
19.2
An experimental cellulase±hemicellulase enzyme mixture from Finnish Sugar Company, Helsinki, Finland.
Number of observations per mean was 6.
c
Mean of FSO2 and FSO2 U.
b
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T. Adogla-Bessa et al. / Animal Feed Science and Technology 82 (1999) 51±61
Fig. 1. Temperature changes as an indication of aerobic deterioration in untreated, enzyme-treated (FS2), urea treated or urea enzyme treated (FS2 U) WCW
harvested and ensiled immediately (IMM) or after 24 h (DEL), with or without evacuation (NOVAC) of air. (& ambient; untreated; * low-enzyme; & medium
enzyme; x high-enzyme; } urea).
T. Adogla-Bessa et al. / Animal Feed Science and Technology 82 (1999) 51±61
57
decreasing (P < 0.05) pH, ensiling DM losses, and contents of butyric acid, NDF, ADF
and cellulose. Such trends were absent in the FSO2 U treated forages. In contrast,
ammonia nitrogen content increased with increasing enzyme application rate in
FSO2 U treated forages but was unaffected by FSO2 treatment.
3.3. Effect of silo sealing treatment
Table 3 shows the effect of the silo sealing treatment on chemical composition and
aerobic stability. Delaying sealing by 24 h significantly reduced (P < 0.01) DM losses
during exposure. Ensiling DM loss was unaffected by sealing treatment in FSO2
treatments, but was lowest (P < 0.05) in silos which were not evacuated after FSO2 U
application.
Table 3
Effect of silo sealing treatment on chemical composition, aerobic stability and fermentation products in WCW
forages conserved with enzyme (FSO2a) and an enzyme±urea mixture (FSO2 U)
Silo sealing treatmentb
Main effects
pH
In vitro organic matter
digestibility (g kgÿ1 DM)
Silo exposure DM loss
(g kgÿ1 DM)
Composition of dry
matter (g kgÿ1 DM)
Total nitrogen
Neutral detergent fibre
Acid detergent fibre
Cellulose
Water soluble
carbohydrates
Fermentation products
Lactic acid
(g kgÿ1 DM)
Acetic acid
(g kgÿ1 DM)
Ammonia-nitrogen
(g kgÿ1 N)
Interaction effects
Ensiling DM loss
(g kgÿ1 DM)
Butyric acid
(g kgÿ1 DM)
a
Immediate
seal evacuation
Immediate
seal-evacuation
24 h
delay evacuation
6.76
570
6.76
590
6.82
570
151
153
124
17.9
477
288
23
79
18.1
451
273
22
88
17.8
484
301
23
80
0.24
16.6
13.3
1.0
3.3
6.1
5.9
3.0
0.54
8.2
7.9
7.8
0.42
231
FSO2
FSO2 U
FSO2
FSO2 U
SEDc
11.0
3.9
2.1
0.1
222
11.0
2.2
2.0
0.1
215
10.9
3.9
1.6
0.1
0.02
14.0
5.0
11.8
0.26
0.12
An experimental cellulase±hemicellulase enzyme mixture from Finnish Sugar Company, Helsinki, Finland.
Mean of FSO2 and FSO2 U treatments.
c
Number of observations per mean was 8.
b
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T. Adogla-Bessa et al. / Animal Feed Science and Technology 82 (1999) 51±61
Seal treatment did not affect N content, in vitro DOMD or structural carbohydrate
contents. An exception was the ADF content which was highest (P < 0.05) when sealing
was delayed for 24 h and lowest (P < 0.05) when silos were not evacuated. WSC content
was higher (P < 0.01) in non-evacuated silos. Delaying silo sealing by 24 h increased pH
(P < 0.01) reduced lactic and butyric acid levels (P < 0.01) compared to the immediately
sealed silos (Table 3). Other fermentation products were unaffected by sealing treatment.
Irrespective of silo seal treatment, untreated and FSO2-treated forages tended to
deteriorate within a week of exposure (Fig. 1) but FSO2 U forages remained stable for
about 30 days. Also enzyme level had no major effect on aerobic stability.
4. Discussion
4.1. Effect of additive type and level
The FSO2 U treatment was better than the FSO2 treatment at degrading cell wall
carbohydrates, enhancing digestibility, minimising DM losses and maintaining aerobic
stability. The presence of urea which as found previously (Deschard et al., 1988; Tetlow,
1992; Adesogan et al., 1998), enhances digestibility and N content and decreases
structural carbohydrate content in WCW, may have contributed to the effectiveness of the
FSO2 U treatment. The urea present would have also promoted alkaline conditions that
inhibit the proliferation of fungi and saccharolytic clostridia that cause aerobic spoilage
and DM losses, respectively (McDonald et al., 1991). Hill and Leaver (1991) and Tetlow
(1992) also reported urea treatment inhibited aerobic deterioration in WCW. In the
rumen, the alkalinity resulting from urea treatment would have buffered rumen acidity
and provided a conducive environment for cellulolysis and growth of rumen bacteria
(Mgheni et al., 1994) and protozoa (Mould and Orskov, 1984). A synchronous supply of
the additional volatile N from the urea and the fermentable energy in the whole crop
forages could have also contributed to the enhancement of microbial growth.
The fact that forages to which FSO2 U was applied had 5±10% lower NDF and ADF
contents than urea-treated forages suggests that the effects of the enzyme and urea were
complimentary. Other authors have also shown that combining urea and cellulase enzyme
treatments resulted in higher digestibility and lower cell wall contents in grass, maize and
sorghum silages than either of the treatments alone (Baintner et al., 1989; Jakhmola et al.,
1990). However, Jacobs (1989) reported reductions in FSO2 activity with increasing pH
and other workers have shown that cellulase±hemicellulase enzymes are most effective at
pH 4.2 (Pitt, 1990). These findings suggest that the alkaline pH resulting from urea
application would inhibit cellulase±hemicellulase enzyme activity. Therefore, the benefits
of combining urea and enzyme treatments in this and previous studies may reflect
differences in the time taken for the treatments to act. While cellulase enzymes can
commence cell wall degradation shortly after they are applied, it can take up to two
weeks for urea application to take effect and substantially increase the pH. The benefits of
the combination of urea and enzyme may therefore reflect an initial degradation of xylans
(hemicellulose) and cellulose by the enzymes followed by further degradation of
lignocellulose bonds in the cell walls (Chesson and érskov, 1984) by the ammonia
T. Adogla-Bessa et al. / Animal Feed Science and Technology 82 (1999) 51±61
59
produced by the urea in the presence of microbial urease. However, increasing enzyme
concentration did not affect the chemical composition of FSO2 U-treated forages in
this study. This suggests that the benefits of the FSO2 U treatment are optimised at the
low level of enzyme application.
In line with the findings of Selmerolsen et al. (1993), increasing the concentration of
the enzyme (when applied alone), increased the WSC content and decreased the ensiling
DM losses, structural carbohydrate and ammonia N contents. These effects which are
probably related to the improved fermentation that often accompanies increasing enzyme
concentration (Woolford, 1984), suggest that the high rate of enzyme application is most
effective for conserving WCW. Indeed the medium and high rates of enzyme application
were comparable or superior to urea treatment at degrading structural carbohydrates and
increasing WSC content, but urea application was consistently superior to the low
enzyme application level.
4.2. Effect of silo sealing treatment
As found previously (Adogla-Bessa and Owen, 1995), exposure DM losses were lower
(P < 0.01) when sealing was delayed for 24 h. This probably reflects the fact that less
volatile constituents were available, and therefore lost from the delayed seal silos due to
the poorer fermentation that occurred. Such poor fermentation in delayed seal silos is due
to the utilisation of WSC by the intrinsic plant enzymes before anaerobiosis prevails
(McDonald et al., 1991). Although this suggests that benefits could arise from wilting
whole crop clamps under practical conditions, the loss in WSC content and attendant
decrease in nutritive value would probably outweigh the benefits of the reduced DM losses.
It is interesting to note that immediate evacuation of silos did not improve the
fermentation or aerobic stability. This suggests that the volume of air trapped in the silo
was not large enough to adversely influence the fermentation process. Thus, the trapped
oxygen was rapidly used up by the post-sealing respiratory process and did not hinder the
establishment of anaerobic conditions (Honig, 1975). It therefore appears that WCW can
be adequately conserved without vacuum evacuation of oxygen in small scale, laboratory
silos. However, this does not minimise the need for adequate compaction and air
exclusion from WCW clamps in practice.
The improvement in aerobic stability resulting from urea application agrees with the
findings of Hill and Leaver (1991) and Adesogan et al. (1996). Tetlow (1983) also
demonstrated that urea treatment of perennial ryegrass decreased the silo temperature
relative to ambient for one month after opening and concluded that urea treatment was
effective against fungi, actinomycetes and yeasts.
It is concluded that increasing the application rate of FSO2 enhanced the fermentation
and stability of WCW. Indeed when applied at 3500 ml tÿ1 DM, FSO2 treatment can be
as effective as urea treatment for conserving WCW harvested at optimal DM yield.
However, urea treatment produces more stable silages with higher protein contents.
Applying a mixture of urea and FSO2 (1750 ml tÿ1 DM) produces greater structural
carbohydrate degradation than urea alone, but the activity of the mixture is reduced when
higher FSO2 concentrations are used. The feeding value of urea and enzyme-treated
WCW were compared in a sequel to this study.
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T. Adogla-Bessa et al. / Animal Feed Science and Technology 82 (1999) 51±61
Acknowledgements
The Finnish Sugar Co. Ltd., Helsinki, Finland is gratefully acknowledged for financing
the study at the institute of Grassland and Environmental Research, Hurley, UK (IGER),
and providing a postgraduate scholarship to Tsatsu Adogla-Bessa. The authors wish to
acknowledge help from personnel of the IGER Hurley in particular R.M. Tetlow and R.D.
Baker who assisted during the initiation and conduct of the experiments. The assistance
of the Department of Applied Statistics and Faculty of Agriculture and Food Analytical
Service of Reading University is also acknowledged.
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Mould, F.L., Orskov, E.R., 1984. Manipulation of rumen fluid pH and its influence on cellulolysis in sacco, dry
matter degradation and the rumen microflora of sheep offered either hay or concentrate. Anim. Feed Sci.
Technol. 16, 1±14.
Pitt, R.E., 1990. Silage and hay preservation, NRAES, New York, 53 p.
Selmerolsen, I., Henderson, A., Robertson, S., McGinn, R., 1993. Cell-wall degrading enzymes for silage 1. The
fermentation of enzyme-treated ryegrass in laboratory silos. Grass and Forage Sci. 48, 45±54.
Smith, D., Groteluschen, R.D., 1966. Carbohydrates in grasses I. Crop Sci. 6, 263±270.
Tetlow, R.M., 1983. The effect of urea on the preservation and digestibility in vitro of perennial ryegrass. Anim.
Feed Sci. Technol. 10, 49±63.
Tetlow, R.M., 1992. A decade of research into whole crop cereals. In: Stark, B.A., Wilkinson, J.M. (Eds.), Whole
Crop Cereals, 2nd ed., Chalcombe Publications, Kent, pp. 1±20.
Tilley, J.M.A., Terry, R.A., 1963. A two-stage technique for the in vitro digestion of silage crops. J. Br. Grassl.
Soc. 18, 104±111.
Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3568±3597.
Wilson, R.F., Wilkins, R.J., 1978. Paraformaldehyde as a silage additive. J. Agric. Sci. 91, 23±29.
Woolford, M.K., 1984. The Silage Fermentation, Marcel Dekker, New York, 350 p.
82 (1999) 51±61
Ensiling of whole crop wheat with
cellulase±hemicellulase based enzymes
3. Comparing effects of urea or enzyme treatment
on forage composition and stability
T. Adogla-Bessaa,1, E. Owena, A.T. Adesoganb,*
a
Department of Agriculture, The University of Reading, Earley Gate,
P.O. Box 236, Reading RG6 6AT, UK
b
Welsh Institute of Rural Studies, University of Wales, Aberystwyth, Llanbadarn Campus,
Aberystwyth SY23 4AL, Wales, UK
Received 21 October 1998; received in revised form 25 June 1999; accepted 20 July 1999
Abstract
This work compared the effect of ensiling technique and application of urea or a cellulase±
hemicellulase enzyme mixture (FSO2) on the conservation of whole crop wheat. Awheat crop harvested
at 600 g dry matter (DM) kgÿ1 was conserved with or without the application of urea (40 g kgÿ1 DM)
and four levels of FSO2 (0, 1750, 3500 and 15 503 ml tÿ1 DM). Each forage (5 kg DM) was conserved
in polythene-bag silos that were evacuated and sealed immediately, sealed immediately but not
evacuated or evacuated and sealed after 24 h. After at least 42 days of conservation, the silos were
opened and analysed for chemical composition, rumen fluid-pepsin in vitro digestibility and aerobic
stability. In non-urea-treated forages, increasing enzyme application rate did not affect in vitro
digestibility, but increased water soluble carbohydrate and lactic acid contents, and reduced pH, neutral
detergent fibre, acid detergent fibre and cellulose contents. Compared to the FSO2 only treatments, urea
treatment increased pH and N content and reduced ensiling DM loss, neutral detergent fibre, acid
detergent fibre, acid detergent lignin and cellulose contents. Application of FSO2 and urea (FSO2 U)
produced forages with higher in vitro digestibility and lower cell wall contents than in FSO2 only
forages. NDF and ADF contents were also 5±10% lower in FSO2 U forages than in those conserved
with only urea. Immediate evacuation of silos did not enhance fermentation quality. Delaying silo
sealing by 24 h increased lactic acid content and aerobic stability relative to either of the immediate seal
treatments. Urea treatment alone and the high enzyme level alone also enhanced aerobic stability.
However, increasing the enzyme application rate in the FSO2 U treatments did not enhance
*
Corresponding author. Tel.: 44-1970-624471; fax: 44-1970-611264
E-mail address: ata@aber.ac.uk (A.T. Adesogan)
1
Present address: University of Botswana, Botswana College of Agriculture, Bag 0027, Gaborone, Botswana.
0377-8401/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 9 9 ) 0 0 1 0 0 - 5
52
T. Adogla-Bessa et al. / Animal Feed Science and Technology 82 (1999) 51±61
stability. These findings indicate that in whole crop wheat, the medium (3500 ml tÿ1 DM) and high
(15 503 ml tÿ1 DM) levels of FSO2 application are as effective as urea in degrading cell walls and
maintaining aerobic stability. However, nutritive value is optimised when whole crop wheat is
conserved using a mixture of urea and the low level (1750 ml tÿ1 DM) of FSO2. # 1999 Elsevier
Science B.V. All rights reserved.
Keywords: Whole crop wheat; Chemical composition; In vitro digestibility; Aerobic stability
1. Introduction
Whole crop wheat (WCW) can be harvested and successfully conserved over a wide
range of crop maturities (350±700 g DM kgÿ1; Adogla-Bessa and Owen, 1995) but the
optimum dry matter (DM) yield is attained at the medium-dough growth stage (550±600 g
DM kgÿ1) (Corral et al., 1977). However, at such advanced growth stages, water soluble
carbohydrate content (WSC) is low and therefore fermentation capacity is reduced. Urea
application has been successfully used to overcome this problem as the resulting alkaline
preservation obviates the need for a high WSC content in the crop. However, several
studies have shown disappointing production results when dairy cows were fed ureatreated WCW harvested at optimal DM yield (Leaver and Hill, 1992). This problem is
thought to relate largely to the lignified pericarp in mature WCW, which prevents digestion
of the grain. Although cellulolytic enzymes have been successfully used to hydrolyse
forage polysaccharides in grass silage (Huhtanen et al., 1985) and whole crop sorghum
silage (Laytimi et al., 1988), little is known about their potential for conserving and
enhancing the nutritive value of WCW. Therefore, this work aimed to determine how
cellulolytic enzymes compare with urea for conserving mature WCW. To this end, the
study evaluated the effect of urea or enzyme treatment on the chemical composition, in
vitro digestibility and aerobic stability of WCW harvested at optimum DM yield. Since
WCW is particularly prone to aerobic deterioration, this work also investigated the effect
of different ensiling techniques on the conservation of WCW. The study is particularly
relevant to the current UK situation where farmers are looking for alternatives to urea
treatment for conserving WCW harvested at optimal DM yield. This is because of the
inefficient utilisation of such forages due to losses of undigested cereal grains in the faeces.
This study follows on from previous work (Adogla-Bessa and Owen, 1995) which
showed that specific cellulase±hemicellulase enzymes enhanced the fermentation of
immature (< 500 g kgÿ1 DM) WCW but did not enhance digestibility or reduce fibre
content. A different enzyme mixture with a higher hemicellulase activity (FSO2) was
investigated in this study.
2. Materials and methods
2.1. Forage preparation and additive treatments
Whole crop winter wheat (cv. `Brock') was cut and harvested with a New Holland 717
metered-chop, forage harvester, at the medium-dough stage (600 g DM kgÿ1). The crop
T. Adogla-Bessa et al. / Animal Feed Science and Technology 82 (1999) 51±61
53
was precision chopped and conserved with an enzyme additive (FSO2, a cellulase±
hemicellulase experimental enzyme mixture from Finnish Sugar, Helsinki, Finland)
applied at rates of 0, 1750 ml tÿ1 DM (low), 3500 ml tÿ1 DM (medium) and 15 503
ml tÿ1 DM (high). Additional treatments included the application of urea (40 g kgÿ1 DM)
alone and the application of a mixture of urea and each of the enzyme levels (FSO2 U).
The enzyme was obtained in liquid form, diluted to a standard volume and applied using
a high velocity pressure sprayer (Kremlin pneumatic sprayer, model J4, Kremlin Spray
Painting Equipment Ltd. Slough, UK). Enzyme activities (IU mlÿ1) for FSO2 were as
follows: cellulase 2.08; carboxymethyl cellulase 54.00; cellobiase 3.24; xylanase 496.9;
(Jacobs, 1989).
Each forage (5 kg) was conserved in a polythene bag silo using the procedure outlined
by Adogla-Bessa and Owen (1995). The silos were evacuated as completely as possible
with a vacuum pump (Edwards Hi-Vac vacuum pump, Crawley, UK) and sealed
immediately, not evacuated and sealed immediately, or evacuated and sealed after 24 h.
2.2. Forage stability measurements
The forages were ensiled for a minimum of 42 days and ensiling DM loss determined
as the weight difference between silo DM content at sealing and opening. Silos were
exposed for 55 days in a controlled environment room (15±178C). Exposure DM loss was
determined as the weight difference between silo DM content at opening and at the end of
the exposure period. Silo temperatures were monitored daily during the exposure period,
as an index of bacterial activity and aerobic deterioration. Thermocouple wires (Ni±Al) were
inserted into the geometric centre of each silo and left in situ. The wires were connected to a
central junction and recording box (Wheatstone Bridge, Comark 1621, Comark Electronics,
Littlehampton, UK) which enabled temperature to be read without disturbing the silo.
2.3. Chemical analyses
Oven DM was determined by drying 400±500 g of fresh or thawed samples at 55±658C
in a forced draught oven for 48 h. A corrected forage DM was determined by the method
of McDonald and Dewar (1960). A glass electrode (WPA pH meter, WPA, Linton, UK)
was used to determine pH on fresh samples. Total nitrogen (N) was determined by complete combustion of freeze-dried samples in a Leco nitrogen analyser (Leco FP-228, Leco
Corporation, St. Joseph, Michigan, USA). Acid detergent fibre (ADF) and neutral
detergent fibre (NDF) were determined by the method of Van Soest et al. (1991); 2.0 ml
of 2% amylase solution was added to aid NDF filtration. WSCs were determined by
measuring the reducing activity of the sugars (Smith and Groteluschen, 1966) in ovendried samples with a Chemlab continuous-flow colorimeter (Chemlab instruments,
Hornchurch, UK). Volatile fatty acids and lactic acid were assessed by gas
chromatography (Wilson and Wilkins, 1978). In vitro digestible organic matter content
(DOMD) was determined by a modification of the 2-stage method of Tilley and Terry
(1963); 10 mg of nitrogen as ammonium sulphate was added at the start of the first stage,
and microbial activity at the end of the first stage was terminated by filtering and adding
acidified pepsin solution to the sample.
54
T. Adogla-Bessa et al. / Animal Feed Science and Technology 82 (1999) 51±61
2.4. Experimental design and statistical analysis
The design was a 2 4 3 factorial arrangement with 2 replicates per treatment. Data
were analysed using the Genstat Statistical Package (Lawes Agricultural Trust, 1988) and
differences between means were tested using the students t-test. F ratios were inspected
and where the main effects were positive and much larger than the interaction effect, they
were also discussed (Mead and Curnow, 1983).
3. Results
3.1. Effect of additive type
Table 1 shows the effect of additive type on forage chemical composition and DM loss.
Silo DM losses during ensiling and exposure were lower (P < 0.01) in forages treated
FSO2 U than in FSO2-treated forages (Table 1). The FSO2 U treatment also gave
higher (P < 0.01) in vitro DOMD and total N contents, and lower (P < 0.05) ADF,
cellulose, ADL and NDF (P < 0.05) contents than the FSO2 treatment. FSO2 U
application also resulted in higher (P < 0.01) pH and acetic acid levels and lower
(P < 0.01) lactic and butyric acid levels. Aerobic spoilage was inhibited for up to 30 days
Table 1
Main effect of enzyme (FSO2) and enzyme urea (FSO2 U) treatment on silo dry matter loss, chemical
composition and fermentation products in WCW forages
SEDb
Additive
pH
In vitro organic matter digestibility
(g kgÿ1 DM)
Silo dry matter loss (g kgÿ1 DM)
Ensiling
Exposure
Composition of dry matter (g kgÿ1 DM)
Total nitrogen
Neutral detergent fibre
Acid detergent fibre
Cellulose
Acid detergent lignin
Fermentation products
Lactic acid (g kgÿ1 DM)
Acetic acid (g kgÿ1 DM)
Butyric acid (g kgÿ1 DM)
Ammonia nitrogen (g kgÿ1 N)
a
b
FSO2a
FSO2 U
4.7
530
8.9
620
11
160
3
126
14.8
492
301
240
52
21.2
441
274
220
49
0.19
13.5
10.9
9
1.8
7.6
5.4
1.9
67
2.4
10.5
0.1
379
0.44
0.34
0.07
10
0.02
11.0
0.2
4.1
An experimental cellulase±hemicellulase enzyme mixture from Finnish Sugar Company, Helsinki, Finland.
Number of observations per mean was 24.
55
T. Adogla-Bessa et al. / Animal Feed Science and Technology 82 (1999) 51±61
by FSO2 U application except at the low enzyme level (Fig. 1). Whereas deterioration
was apparent within 10 days in the FSO2-treated forages.
Compared to the FSO2-treated forages, urea treatment increased pH and N content and
reduced ensiling DM loss, NDF, ADF, ADL and cellulose contents (Table 2).
3.2. Effect of enzyme application rate and interaction with additive type
Table 2 shows the effect of enzyme concentration on chemical composition. Increasing
the rate of enzyme application did not affect in vitro DOMD or exposure DM losses. In
FSO2-treated forages, increasing enzyme concentration increased WSC content whilst
Table 2
Effect of enzyme (FSO2a) application rate on chemical composition, in vitro digestibility and fermentation
products in WCW forages conserved with enzymes or with an enzyme±urea mixture (FSO2 U)
SEDb
Enzyme application rate (ml tÿ1 DM)
0
(untreated)
1750
(low)
3500
(medium)
15 503
(high)
153
137
143
138
4.7
8.0
570
4.4
8.1
570
4.3
7.6
580
6.6
8.2
580
0.63
0.42
16.0
FSO2
FSO2 U
FSO2
FSO2 U
11.9
2.8
4.9
8.8
11.4
3.4
4.8
8.6
11.1
3.2
4.7
8.9
9.4
4.1
4.4
8.8
0.31
Total nitrogen
(g kgÿ1 DM)
Neutral detergent fibre
(g kgÿ1 DM)
Acid detergent fibre
(g kgÿ1 DM)
Cellulose
(g kgÿ1 DM)
FSO2
FSO2 U
FSO2
FSO2 U
FSO2
FSO2 U
FSO2
FSO2 U
14.5
21.2
526
448
333
276
250
22
14.3
21.4
526
461
320
262
250
21
14.7
21.3
485
418
288
256
240
20
15.6
20.7
432
469
263
301
200
23
0.39
Water soluble carbohydrates
(g kgÿ1 DM)
Butyric acid
(g kgÿ1 DM)
FSO2
FSO2U
FSO2
FSO2 U
64
77
2.5
0.1
69
82
2.0
0.1
89
82
1.7
0.1
118
76
1.9
0.1
FSO2
FSO2 U
74
87
Main effectsc
Silo dry matter loss during
exposure (g kgÿ1 DM)
Lactic acid
Acetic acid
Digestible organic matter
(g kgÿ1 DM)
Interaction effectsb
Ensiling DM loss (g kgÿ1 DM)
pH
Ammonia-nitrogen (g kgÿ1 N)
a
79
446
57
487
57
498
5.8
0.03
27.1
21.8
17.0
5.3
0.14
19.2
An experimental cellulase±hemicellulase enzyme mixture from Finnish Sugar Company, Helsinki, Finland.
Number of observations per mean was 6.
c
Mean of FSO2 and FSO2 U.
b
56
T. Adogla-Bessa et al. / Animal Feed Science and Technology 82 (1999) 51±61
Fig. 1. Temperature changes as an indication of aerobic deterioration in untreated, enzyme-treated (FS2), urea treated or urea enzyme treated (FS2 U) WCW
harvested and ensiled immediately (IMM) or after 24 h (DEL), with or without evacuation (NOVAC) of air. (& ambient; untreated; * low-enzyme; & medium
enzyme; x high-enzyme; } urea).
T. Adogla-Bessa et al. / Animal Feed Science and Technology 82 (1999) 51±61
57
decreasing (P < 0.05) pH, ensiling DM losses, and contents of butyric acid, NDF, ADF
and cellulose. Such trends were absent in the FSO2 U treated forages. In contrast,
ammonia nitrogen content increased with increasing enzyme application rate in
FSO2 U treated forages but was unaffected by FSO2 treatment.
3.3. Effect of silo sealing treatment
Table 3 shows the effect of the silo sealing treatment on chemical composition and
aerobic stability. Delaying sealing by 24 h significantly reduced (P < 0.01) DM losses
during exposure. Ensiling DM loss was unaffected by sealing treatment in FSO2
treatments, but was lowest (P < 0.05) in silos which were not evacuated after FSO2 U
application.
Table 3
Effect of silo sealing treatment on chemical composition, aerobic stability and fermentation products in WCW
forages conserved with enzyme (FSO2a) and an enzyme±urea mixture (FSO2 U)
Silo sealing treatmentb
Main effects
pH
In vitro organic matter
digestibility (g kgÿ1 DM)
Silo exposure DM loss
(g kgÿ1 DM)
Composition of dry
matter (g kgÿ1 DM)
Total nitrogen
Neutral detergent fibre
Acid detergent fibre
Cellulose
Water soluble
carbohydrates
Fermentation products
Lactic acid
(g kgÿ1 DM)
Acetic acid
(g kgÿ1 DM)
Ammonia-nitrogen
(g kgÿ1 N)
Interaction effects
Ensiling DM loss
(g kgÿ1 DM)
Butyric acid
(g kgÿ1 DM)
a
Immediate
seal evacuation
Immediate
seal-evacuation
24 h
delay evacuation
6.76
570
6.76
590
6.82
570
151
153
124
17.9
477
288
23
79
18.1
451
273
22
88
17.8
484
301
23
80
0.24
16.6
13.3
1.0
3.3
6.1
5.9
3.0
0.54
8.2
7.9
7.8
0.42
231
FSO2
FSO2 U
FSO2
FSO2 U
SEDc
11.0
3.9
2.1
0.1
222
11.0
2.2
2.0
0.1
215
10.9
3.9
1.6
0.1
0.02
14.0
5.0
11.8
0.26
0.12
An experimental cellulase±hemicellulase enzyme mixture from Finnish Sugar Company, Helsinki, Finland.
Mean of FSO2 and FSO2 U treatments.
c
Number of observations per mean was 8.
b
58
T. Adogla-Bessa et al. / Animal Feed Science and Technology 82 (1999) 51±61
Seal treatment did not affect N content, in vitro DOMD or structural carbohydrate
contents. An exception was the ADF content which was highest (P < 0.05) when sealing
was delayed for 24 h and lowest (P < 0.05) when silos were not evacuated. WSC content
was higher (P < 0.01) in non-evacuated silos. Delaying silo sealing by 24 h increased pH
(P < 0.01) reduced lactic and butyric acid levels (P < 0.01) compared to the immediately
sealed silos (Table 3). Other fermentation products were unaffected by sealing treatment.
Irrespective of silo seal treatment, untreated and FSO2-treated forages tended to
deteriorate within a week of exposure (Fig. 1) but FSO2 U forages remained stable for
about 30 days. Also enzyme level had no major effect on aerobic stability.
4. Discussion
4.1. Effect of additive type and level
The FSO2 U treatment was better than the FSO2 treatment at degrading cell wall
carbohydrates, enhancing digestibility, minimising DM losses and maintaining aerobic
stability. The presence of urea which as found previously (Deschard et al., 1988; Tetlow,
1992; Adesogan et al., 1998), enhances digestibility and N content and decreases
structural carbohydrate content in WCW, may have contributed to the effectiveness of the
FSO2 U treatment. The urea present would have also promoted alkaline conditions that
inhibit the proliferation of fungi and saccharolytic clostridia that cause aerobic spoilage
and DM losses, respectively (McDonald et al., 1991). Hill and Leaver (1991) and Tetlow
(1992) also reported urea treatment inhibited aerobic deterioration in WCW. In the
rumen, the alkalinity resulting from urea treatment would have buffered rumen acidity
and provided a conducive environment for cellulolysis and growth of rumen bacteria
(Mgheni et al., 1994) and protozoa (Mould and Orskov, 1984). A synchronous supply of
the additional volatile N from the urea and the fermentable energy in the whole crop
forages could have also contributed to the enhancement of microbial growth.
The fact that forages to which FSO2 U was applied had 5±10% lower NDF and ADF
contents than urea-treated forages suggests that the effects of the enzyme and urea were
complimentary. Other authors have also shown that combining urea and cellulase enzyme
treatments resulted in higher digestibility and lower cell wall contents in grass, maize and
sorghum silages than either of the treatments alone (Baintner et al., 1989; Jakhmola et al.,
1990). However, Jacobs (1989) reported reductions in FSO2 activity with increasing pH
and other workers have shown that cellulase±hemicellulase enzymes are most effective at
pH 4.2 (Pitt, 1990). These findings suggest that the alkaline pH resulting from urea
application would inhibit cellulase±hemicellulase enzyme activity. Therefore, the benefits
of combining urea and enzyme treatments in this and previous studies may reflect
differences in the time taken for the treatments to act. While cellulase enzymes can
commence cell wall degradation shortly after they are applied, it can take up to two
weeks for urea application to take effect and substantially increase the pH. The benefits of
the combination of urea and enzyme may therefore reflect an initial degradation of xylans
(hemicellulose) and cellulose by the enzymes followed by further degradation of
lignocellulose bonds in the cell walls (Chesson and érskov, 1984) by the ammonia
T. Adogla-Bessa et al. / Animal Feed Science and Technology 82 (1999) 51±61
59
produced by the urea in the presence of microbial urease. However, increasing enzyme
concentration did not affect the chemical composition of FSO2 U-treated forages in
this study. This suggests that the benefits of the FSO2 U treatment are optimised at the
low level of enzyme application.
In line with the findings of Selmerolsen et al. (1993), increasing the concentration of
the enzyme (when applied alone), increased the WSC content and decreased the ensiling
DM losses, structural carbohydrate and ammonia N contents. These effects which are
probably related to the improved fermentation that often accompanies increasing enzyme
concentration (Woolford, 1984), suggest that the high rate of enzyme application is most
effective for conserving WCW. Indeed the medium and high rates of enzyme application
were comparable or superior to urea treatment at degrading structural carbohydrates and
increasing WSC content, but urea application was consistently superior to the low
enzyme application level.
4.2. Effect of silo sealing treatment
As found previously (Adogla-Bessa and Owen, 1995), exposure DM losses were lower
(P < 0.01) when sealing was delayed for 24 h. This probably reflects the fact that less
volatile constituents were available, and therefore lost from the delayed seal silos due to
the poorer fermentation that occurred. Such poor fermentation in delayed seal silos is due
to the utilisation of WSC by the intrinsic plant enzymes before anaerobiosis prevails
(McDonald et al., 1991). Although this suggests that benefits could arise from wilting
whole crop clamps under practical conditions, the loss in WSC content and attendant
decrease in nutritive value would probably outweigh the benefits of the reduced DM losses.
It is interesting to note that immediate evacuation of silos did not improve the
fermentation or aerobic stability. This suggests that the volume of air trapped in the silo
was not large enough to adversely influence the fermentation process. Thus, the trapped
oxygen was rapidly used up by the post-sealing respiratory process and did not hinder the
establishment of anaerobic conditions (Honig, 1975). It therefore appears that WCW can
be adequately conserved without vacuum evacuation of oxygen in small scale, laboratory
silos. However, this does not minimise the need for adequate compaction and air
exclusion from WCW clamps in practice.
The improvement in aerobic stability resulting from urea application agrees with the
findings of Hill and Leaver (1991) and Adesogan et al. (1996). Tetlow (1983) also
demonstrated that urea treatment of perennial ryegrass decreased the silo temperature
relative to ambient for one month after opening and concluded that urea treatment was
effective against fungi, actinomycetes and yeasts.
It is concluded that increasing the application rate of FSO2 enhanced the fermentation
and stability of WCW. Indeed when applied at 3500 ml tÿ1 DM, FSO2 treatment can be
as effective as urea treatment for conserving WCW harvested at optimal DM yield.
However, urea treatment produces more stable silages with higher protein contents.
Applying a mixture of urea and FSO2 (1750 ml tÿ1 DM) produces greater structural
carbohydrate degradation than urea alone, but the activity of the mixture is reduced when
higher FSO2 concentrations are used. The feeding value of urea and enzyme-treated
WCW were compared in a sequel to this study.
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T. Adogla-Bessa et al. / Animal Feed Science and Technology 82 (1999) 51±61
Acknowledgements
The Finnish Sugar Co. Ltd., Helsinki, Finland is gratefully acknowledged for financing
the study at the institute of Grassland and Environmental Research, Hurley, UK (IGER),
and providing a postgraduate scholarship to Tsatsu Adogla-Bessa. The authors wish to
acknowledge help from personnel of the IGER Hurley in particular R.M. Tetlow and R.D.
Baker who assisted during the initiation and conduct of the experiments. The assistance
of the Department of Applied Statistics and Faculty of Agriculture and Food Analytical
Service of Reading University is also acknowledged.
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