The Effects of Different Additives on Silage Gas Production, Fermantation Kinetics and Silage Quality

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Ozean Journal of Applied Sciences 2(1), 2009
11
The Effects of Different Additives on Silage Gas Production, Fermantation Kinetics
and Silage Quality
B. Zehra Sariçiçek and Ünal KILIÇ
Department of Animal Science of Agricultural Faculty of Ondokuzmayıs University, Samsun, Turkey
E-mail address:bzehra@omu.edu.tr
______________________________________________________________________________________
Abstract: The objective of study was to explain the effects of differ additives on the nutrient, energy
content, gas production and parameters, fermentation kinetics and silage quality of corn silage. Test
silages from fresh material, wilted for 6 hours were conserved into 1.5-litre jars and opened for analysis in
60 days. Additives (additive ratio) were wheat ground (5%), formic acid (2.5 l/ton), urea (1%), chemical
material, (Artturi Imarın Virtanen; AIV) (8%), biological material (L. Plantarum + P.acidilactici +
L.salivarus) (10 g/ton). Gas production (GP) and its parameters were studied by using in in vitro gas
production technique with ruminally fistulated Sakız x Karayaka rams. Silage treated with wheat ground
had significantly greater OM, CP content, TE and GP compared with other groubs (P<0.01). Ash content
of silage was the highest in silage treated with AIV (P<0.01). The pH value after 96 h. of incubation was
lewer in silage treated with inoculant compared with AIV (P<0.05). The silage added AIV decreased the
ME, NEL and OMD (P<0.01). While lactic acid concentration were the highest in silage treated with wheat
and inoculant, it was the lowest in silage treated with urea (P<0.01). Silage NH3-N and butyric acid
concentration were significantly higher in silage treated with urea (P<0.01).
The highest flieg point was obtained from treatment with AIV.
Keywords: corn silage, additives, gas production, silage quality
______________________________________________________________________________________
INTRODUCTION
Considering the real climate conditions, silage is the best method for preserving fresh forage with minimal
losses. Silage quality and nutritional value are influenced by numerous biological and technological factors,
When the proper ensilage techniques are used, silage will have a high nutritive value and hygienic quality.
However, the results in practice indicate that the quality of silage is often poor or even unsatisfactory.
These results are usually achieved when the fermentation condition are difficult. (Lattemae et al., 2006).
Factors which influence fermentation include degree of gren fodder wilting, length of cut, ensiling
technology type, and amount of an additive used (Haigh, 1988). Silage additives include feedstuffs, urea,
inoculants and acids. (Weiss and Underwood, 2006). The major goal in silage making is to preserve silage
material with minimum nutrient loss. In order to achieve this goal, growth of lactic acid bacteria should be
stimulated. Especially, formic acid is widely used to accomplish this target. While wheat is commonly used
to provide readily available energy for lactic acid fermentation, bacterial inoculant is used to establish a
desirable microbial flora in silage.
Corn has low concentrations of protein and some minerals, but high concentrations of fermentable
carbohydrates. Nutritional additives such as urea however, are benefical. The justifications for using non
protein nitrogen (NPN) have been prolonged aerobic stability during the feedout phase and the addition of
an economical nitrogen source to low protein crops, such as corn (Weiss and Underwood, 2006).
Feed evaluation for ruminant is often based on an estimate of rumen digestibility, even though such
measurements alone cannot predict how ruminants will utilise feed nutrients. However, in vivo digestibility
depends on a range of factors, including potential digestibility, rate of digestion, fractional rumen outflow
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and efficiency of microbial production. Gas production techniques are used in many laboratories to study
fermentation kinetics of ruminant feeds. In vitro gas production system (Menke et al., 1979; Mauricio et al.,
2001) provide and estimate of dry matter (DM) and OM digestion anda re an indicator of the end products
produced. (Bueno et al., 2005). Maize silage is an important component of ruminant diets and its energy
value is widely estimated from chemical composition and in vitro organic matter digestibility (OMD)
(DeBoever et al., 2005). In this study, the effect of different silage additive added during ensiling period
was investigated on the nutrient, the silages quality, gas production, gas production parameters, energy
values, organic matter digestibility of maize silage.
MATERIALS AND METHODS
Maize (Arifiye) as a silage material was used in this study. As additives; wheat ground, urea (contains 46%
N), formic acid, AIV solution (composed 1 part H2SO4, 1 part H2SO4, 1 part HCl and 6 part water),
microbial inoculant (Maize-All® GS obtained from Alltech-Pioneer). Maize-all GS is formulated with
three lactic acid-producing bacteria (Lactobacillus plantarum, Pediococcus acidilactici and Lactobacillus
salivarus) and high level amylase.
In this study, maize (arifiye) crop was grown at the Agricultural Faculty Experimental Farm of
Ondokuzmayıs University, Samsun. It was harvested by a one row forage harvester at dough stage of
kernel maturuty. The fresh material which will be ensilaged was chopped to about 2 cm length and were
ensiled in laboratory type 2.0-l glass jars, equipped with a lid that enable gas release only. The maize from
dough stage was wilted for 24 h in the field, to about 35%DM, before ensiling. Six different silages were
prepared (quadriplicate) form chopped forage. Silage treatments included control (no additives) (CS). 5%
wheat ground (WGS), 2.5 lt/ton formic acid (FS), 10 g/t microbial inoculant (Maize-All® GS). As
recommend by the manufacturer, inoculant was added at 1.0x1011 cfu/g of fresh forage (MAS), 1% urea
(US) and 8% AIV (AIVS). They were stored for 60 d in the dark room with a temparature ranging between
20 to 25 oC. At the end of the ensiling period (60 d), the silages were opened. Representative samples were
dried at 48 ˚C in a forced-air oven for 72 hours.
After drying, silage samples were ground through a 1-mm screen for chemical analysis. The dry matter
(DM) (105 oC), ash (550 oC), crude protein (CP)(Kjeldahl Nx6.25), ether extract (EE), crude fiber (CF) of
the silages were determined by the methods described by AOAC (1990). Volatile fatty acid and NH3-N
contents in rumen fluid were determined using Markham Steam Distillation procedure (Markham, 1942)
(Table 1).
All silage organic acids analysis was accomplished by using gas chromatograph (Shimadzu, GC-14B) as
described by Leventini et al. (1990). Furthermore, quality analysis (Kilic, 1986), were made in silages, and
nitrogen free extract (NFE) was determined by calculation. All cemical analysis was carried out in
triplicate.
Table 1. pH, TVFA and NH3-N content of rumen fluid
pH TVFA (mmol/l) NH
3
-N (mg/l)
Average 6.20 119 310
Lowest 5.92 90 280
Highest 6.29 136 427
TVFA:Total volatile fat acid
The pH of each sample was determined in triplicate using approximately 25 g wet ensilage added to 100 ml
of distilied water. After hydration for 10 min using blender, pH was determined using digital pH meter.
Flieg points were calculated in silages according to Kılıc (1986) (Flieg Point = 220+(2 x % Dry Matter - 15)
– 40 x pH).
Three Sakız x Karayaka rams aged 2 with ruminal cannulas were used in gas production technique. Rumen
fluid was obtained from three fistulated sheep fed twice daily (08.30-16.30) with a diet containing grass
hay (%60) and concentrate (%40) (Table 2). The samples (milled through a 1-mm sieve) were incubated in

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vitro rumen fluid in calibrated glass syringes following the procedures of Menke et al. (1979), Menke and
Steingass (1988) and Blümmel and Ørskov (1993). Approximately 200 mg dry weight of sample was
weighed in triplicate into calibrated glass syringes of 100 ml. Rumen fluid was collected before the
morning feeding. The syringes were prewarmed at 39 ºC before the injection of 30 ml Rumen-buffer
mixture consisting of 10 ml rumen fluid and 20 ml digestion medium into each syringe followed by
incubation in a water bath at 39 ºC. Triplicates of each sample were used in two separate runs. Readings of
gas production recorded before incubation and 3, 6, 9, 12, 24, 48, 72 and 96 h after incubation. Total gas
values corrected for blank incubation. Cumulative gas production data were fitted to the model of Ørskov
and McDonald (1979) by NEWAY computer package programme. y = a+b(1-e-ct)
Table 2. Chemical composition of silages (% DM)
Feed E E Ash CP CF NFE
Control Mean 2,09
ab
6,93
b
7,05
b
22,07
a
61,85
b
Wheat Mean 1,65
ab
5,18
c
8,32
a
19,49
b
65,37
a
Formic acid Mean 2,25
a
5,65
bc
7,22
b
21,03
a
63,84
ab
Maize-all Mean 2,20
ab
5,64
bc
7,16
b
22,24
a
62,76
b
Urea Mean 2,07
ab
6,57
bc
12,10
a
22,78
a
56,48
c
AIV Mean 1,60
b
10,22
a
7,19
b
22,19
a
58,81
b
SEM 0,083 0,361 0,1 10 0,254 0,449
Significant ** ** ** ** **
EE: Eter extract, CP:crude protein, CF:Crude fiber, NFE:Nitrogen free extract
a,b,..:Means with different superscript within same column significantly differ (P<0.05).
** (P<0.01), * (P<0.05)
Where; a: the gas production from the immediately soluble fraction (ml), b: the gas production from the
insoluble fraction (ml), c: the gas production rate constant for the insoluble fraction (ml/h), a+b: potential
gas production (ml), t: incubation time (h), y: gas produced at time “t”. Oganic matter digestibility (OMD),
metabolisable energy (ME) (Menke et al. 1979) and NEL (Menke and Steingass,1988) contents of forages
were estimated using equations given below:
OMD, % = 14.88+ 0.889 GP + 0.45 CP + 0.065 A, Where; GP:24 h net gas production (ml/200mg DM),
CP:Crude protein (%), A:Ash content (%)
ME, (MJ/kg DM) = 2.20+0.136 GP + 0.0574 CP
NEL, (MJ/kg DM) = 0.101 GP + 0.051 CP + 0.112 EE
Where; GP: 24 h net gas production (ml/200mg DM), CP: g/kg DM, EE: g/kg DM
One-way analysis of variance (ANOVA) was carried out to compare gas production, gas production
parameters, Energy values, DMD and OMD values using General Linear Model (GLM) of SPSS 12.0
package programs. Significance between individual means were identified using the Duncan’s multiple
range test.
RESULTS AND DISCUSSION
Total volatile fat acids (TVFA), pH and NH3-N of rumen liquid were given Table 1. Chemical composition
of silages are presented in Table 2. Results of gas production and gas production parameters, organic
matters digestibility (OMD), ME and NEL were given in Table 3. Organic acids concentration, pH, NH3-N
and Flieg point of silages were given Table 4.
According to the results of this study, the TVFA, pH and NH3-N content of rumen liquid for in vitro gas
production technique were normal level (McDonald et al.,1996; Menke and Steingass,1988) The highest
DM content was found for silage treated with wheat and the lowest was obtained from silage treated with
AIV.

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Table 3. Gas production and gas production parameters, estimated energy content and organic matter
digestibility of silages
Gas
production
Control Wheat Formic
acid
Maize
all
Urea Aiv SEM Sig
3 7,99 9,81 7,05 8,22 7,27 10,12 0,468 NS
6 13,06 16,01 12,31 13,39 11,75 12,82 0,617 NS
9 17,64 21,44 16,99 17,97 15,75a 15,24 0,800 NS
12 21,77
ab
26,18
a
21,16
ab
22,01
ab
19,30
ab
17,40
b
0,970 **
24 34,61
ab
39,91
a
33,78
bc
34,11
ab
30,03
bc
24,10
c
1,427 **
48 48,78
ab
52,86
a
46,95
ab
46,38
ab
41,05
b
31,49
c
1,742 **
72 55,08
a
57,55
a
52,45
ab
51,33
ab
45,43
b
35,01
c
1,793 **
96 57,91
a
59,30
a
54,80
a
53,40
ab
47,19
b
36,80
c
1,787 **
pH 6,73
ab
6,61
ab
6,70
ab
6,59
b
6,69
ab
6,74
a
0,019 *
Gas production parameters
a, ml 2,36
b
2,69
b
1,13
b
2,38
b
2,23
b
7,11
a
0,491 **
b, ml 57,94
a
57,71
a
55,51
a
52,67
a
46,14
b
32,44
c
1,982 **
c, ml/h 0,04 0,05 0,04 0,04 0,04 0,04 0,001 NS
RSD 2,04 2,40 1,67 1,52 1,45 1,34 0.219 NS
Energy and OMD
ME,
MJ/kg
DM 7,31a 8,10a 7,21a 7,25a
6,98ab 5,89b
1.193
**
NEL,
MJ/kgDM 4,09a 4,64a 4,03a 4,06a
3,88a 2,98b
1.145
**
OMD, % 49,27
a
54,44
a
48,53
a
48,79
a
47,45
ab
40,21
b
1. 253 **
a,b,..:Means with different superscript within same column significantly differ (P<0.05).
** (P<0.01), * (P<0.05)
NS:Not significant
Table 4. Organic acid, pH, NH3-N concentration and estimated Flieg point of silage
Control Wheat Formic
acid
Maize-
all
Urea AIV SEM Significant
pH 3.55a 3.45a 3.70aa 3.55a 3.65a 1.55b 0.35 **
DM, % 26.17b 28.74a 25.17d 26.22b 26.08c 17.69e 0.64 **
Laktic
acid
2.97b 3.60a 3.09b 3.46a 1.66d 2.09c 0.44 **
Acetic
acid
1.28b 1.05b 2.19a 1.68b 1.26b 2.12a 0.48 **
Butyric
acid
- 0.004 - 0.003 0.05 - 0.43 *
NH3-N
g/kgDM
0.90b 1.26ab 0.78b 0.90b 2.55a 1.83ab 0.38 **
Flieg
point
115.36 124.48 107.30 115.41 111.15 178.38
Quality Very
good
Very
good
Very
good
Very
good
Very
good
Very
good
a,b,..:Means with different superscript within same column significantly differ (P<0.05).
** (P<0.01), * (P<0.05)
There were significant differences between the AIV and WGS (P<0.01). Weiss and Underwood (2006)
reported that addition of wheat into silage increased DM content of wet silage, which underlines our result.
The highest EE was found for FAS (P<0.01) and the lowest was obtained from AIVS (P<0.01).

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While addition of AIV into silage increased, addition of wheat ground decreased crude ash content of
silage. There were significant differences between the AIV and WGS treatments (P<0.01). The results
obtained in the present experiment are supported by Olt et al. (2005), who reported that AIV is a rich
mineral acid source.
CP content of silage were the significantly improved by treatment with urea and wheat ground (P<0.01).
The highest increase was obtained with addition urea. Bolsen et al. (1996) reported that urea is an
economical nitrogen source to low protein crops, such as corn. Many researchers have reported that the
urea addition increased the CP content of silage (Bolsen et al., 1992).
The lowest cellulose content of silage was found for silage treated with AIV. The differences between the
AIVS and the other groups were significant (P<0.01). This decrease in cellulose concentration may have
resulted from increased cell wall degradability due to increased silage fermentation caused by addition of
chemical additive AIV. This result is consistent with Olt et al. (2006), who reported that cellulose content
declined in silage treated with AIV. Cellulose content of silage were not affected by the other additives.
While the highest N free extract (NFE) content was obtained from silage treated with WG, and the lowest
for the silage treated with urea. The differences between the WGS and the other groubs (CS, MAS, US and
AIVS) were significant (P<0.01). This increase in NFE content for WS could be explained with the fact,
that wheal is a rich carbohydrate source. This statement was in agreement with the reporting of Weiss and
Underwood (2006). On the other hand, decrease in NFE for US may has resulted of a rich nitrogen source
of urea.
As seen in Table 3, gas production of silages were not affected by treatments for incubation after the first 3,
6 and 9 hour, but silage treated with wheat ground for 12, 24, 48, 72 and 96 h. incubation were numerically
higher than that of control silage. This could be due to the fact that the grain (wheat) is a relatively rich
source of easily fermentable carbohydrate. The lowest gas production were obtained from AIV and urea
treatment for 72 and 96 h incubation. The differences between the AIV and the other groups were
significant (P<0.01). Gas production values of CS were in agreement with the findings of Kılıç and
Sarıçiçek (2005) and Mould et al. (2000) reported that gas production for 96 h. was 53.-56.0 ml/200ml OM,
what also supports our result.
The highest rumen pH value was found for AIVS after 96 h and the lowest for silage treated with Maize-all
(MAS). AIVS was significantly different from the MAS (P<0.01). The pH value of AIVS in present study
was higher than the findings of Mertens et al. (1997). The highest “a” value was obtained from silage
treated with AIV and the lowest for FAS, but the other additives did not affect the a value. While decrease
in formic acid treated group can be explained with limited degradability coused by formic acid; on the other
hand, an increase can also be explained with increased degradability in the AIV treated group due to the
very strong acid content of AIV. A value obtained in the present experiment was higher than the
finding(1.68 ml) reported by Kılıç and Sarıçiçek (2005), contrary, the a value for corn silage in this study
was lower than findings (7.4 ml) of Compos et al. (2000). These discrepancies may be attributed to
differences in varieties of these corn silages and differences in sample preparation prior to incubation
among the studies.
The highest “b” value was found for CS (untreated), but the lowest “b” value were obtained from AIVS and
US. The AIVS group were significantly different from the other groups (P<0.01). There were no
differences between the silages in terms of “c” value (rate of gas production). The result obtained in the
present experiment are supported by Compos et al. (2000), who reported that c value of corn silage was
0.04 ml.
The lowest ME, NEL and OMD between the silages were obtained from the silage treated with AIV. AIVS
group were significantly different from control and the other groups (P<0.01). Olt et al. (2005) suggested
that OMD of silage could be affected by chemical acids such as AIV treatments. The ME, NEL and OMD
of silage treated with wheat ground was numerically higher than the control group.

Ozean Journal of Applied Sciences 2(1), 2009
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The current study showed similarities with the reports of Hamilton (2005) in that ME for corn silage was
between 5-13 MJ/kg DM. The ME of corn silage in this study was lower than the findings reported by
Getachew et al. (2002) (8.0-9.35 MJ/kg DM), which is near result to silage treated with wheat ground.
In this study, the NEL of corn silage (untreated) are in accordance with those (4.72 MJ/kg DM) reported by
Kılıç and Sarıçiçek (2005).
While OMD value of CS was obtained by Lee et al. (2000) as 79.60%, by Getachew et al. (2002) as 53.55-
62.35% and by Sarıçiçek and Çayıroğlu (2001) as 53.70 %. The result of WGS were close to the finding of
Getachew et al. (2002). Nocek and Tamminga (1991) reported that, ruminal starch degradability of wheat
was higher than barley, sorghum and corn, and that wheat and corn starch degradability were 80-90 % and
55-70 %, respectively.
The lowest pH value was found for AIVS. The differences between the AIVS and the other groups were
significant (P<0.01). The decrease in pH could be attributed to the strong mineral acid content of AIV.
Ludenmark (2005) reported that AIV additive into silage decreased the pH value, and the best silages
qualities were achieved with the additive AIV.
The highest lactic acid content were found for WGS and MAS (P<0.01). This could be due to the fact that
the wheats are relatively rich source of carbohydrate, which is easily fermentable. On the other hand, many
rearchers reported that bacterial inoculants have positive effects on pH and lactic acid levels, an indication
of goal fermentation (Bolsen et al., 1992; Handerson, 1993; Weiss and Underwood, 2006).
The lowest lactic acid concentration was found for US. Lactic acid content for US were significantly
different from the WGS, MAS and the other groubs (P<0.01). Urea is a rich nitrogen source. This could be
due to the fact the urea is a rich source of nitrogen. Silage treated with formic acid showed numerically
greater lactic acid concentrations compared with control. Kennedy (1990) has reported that addition of
formic acid into silage decreased silage lactic acid content by limiting silage fermentation, but also
Charmly et al. (1990) reported that formic acid increased silage lactic acid concentration.
While addition of wheat ground into silage numerically decreased, addition of formic acid and AIV
increased silage acetic acid concentration (P<0.01). Some researchers reported a decrease; other reported
no changes in concentrations of acetic acid with addition of formic acid and AIV into silage (O’Kiely et al.,
1990; Kennedy, 1990).
While addition of urea into silage increased butyric acid concentration (P<0.05), but were not affected by
the other treatments. The highest NH3-N cocentration was found for US. The differences between the urea
treated group and formic acid, maize-all groub were significant (P<0.01). These results are consistent with
Bolsen et al. (1996), who reported that N content of silage increased treatment with urea.
In this study, the flieg point of silages ranged from 107.3 to 178.38. Although no statistical analysis of
silage flieg point was conducted, the following trends were apparent. The AIVS and WGS had the highest
point among the silages. The pH value of silage decreased by AIV treatment and DM content of silage
increased by wheat treatment. This leads to the increase in the flieg point.
The effect of AIV, formic acid, urea, inoculant and wheat ground treatments on corn silage were
investigated in this study. The results showed, that silage additives had positive and/or negative effect on
nutrient, silage fermentation, OMD, energy content, gas production and gas production parameters and
quality of silage. However, effects of these additives on silage fermentation, gas production, gas production
parameters, OMD and silage quality should further be studied to determine proper silage additive.
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