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Silage extracts used to study the mode of action of silage
inoculants in ruminants

Richard E. Muck1*, Zwi G. Weinberg2 and Francisco E. Contreras-Govea3

1 USDA, Agricultural Research Service, US Dairy Forage Research Center, 1925 Linden Drive, Madison, Wisconsin,
53706 United States

2 The Volcani Center, Bet Dagan, Israel
3 University of Wisconsin-Madison, Madison, Wisconsin, United States

*e-mail: richard.muck@ars.usda.gov

Lucerne and two maize crops were ensiled with and without Lactobacillus plantarum and fermented for 4 or 60 d
to assess the effect of inoculant on in vitro rumen fermentation of the resulting silages. Water and 80% ethanol ex-
tracts of the silages were also analysed for effects on in vitro rumen fermentation. The inoculant affected lucerne
silage characteristics but had little effect on the maize silages. In vitro fermentation of the silages showed few ef-
fects except increased microbial biomass yield (MBY) at 24 h in the inoculant-treated lucerne silages. In vitro fer-
mentation of the lucerne silage water extracts produced no differences due to treatment except for reduced MBY
in the inoculant-treated extracts. The ethanol extracts produced results inconsistent with the in vitro results of the
silages. Consequently it appears that the factor in in vitro fermentation of inoculated silages causing increased MBY
was in neither the water nor ethanol extracts.

Key words: inoculant, in vitro fermentation, silage, microbial biomass yield, gas production

Introduction

Microbial silage inoculants are additives used to improve silage fermentation (Muck and Kung 1997). These ad-
ditives also may increase milk production or daily gain in livestock, but the mechanisms are unknown (Weinberg
and Muck 1996, Kung and Muck 1997). The most common silage inoculants contain facultative heterofermentative
lactic acid bacteria (LAB) that shift fermentation toward lactic acid production, reducing acetic acid and ethanol.
Based on analysis of data from 47 experiments, a 10 g kg-1 DM increase in lactic acid and 10 g kg-1 DM reduction
in acetic acid should increase energy-corrected milk by 0.12 kg d-1 (Huhtanen et al. 2003). Inoculants often reduce
ammonia in silages. That is also correlated to higher milk production; a 10 g kg-1 N reduction in ammonia would
be expected to increase energy-corrected milk by 0.19 kg d-1 (Huhtanen et al. 2003). Sometime inoculants reduce
the amount of fermentation products, and that should have a positive effect on milk production (Huhtanen et al.
2003, Jaakkola et al. 2006). Unfortunately, the expected improvement in milk production from all of these shifts
in silage composition are much less than the observed average improvement in milk production (1.4 kg d-1) from
feeding inoculated silage (Kung and Muck 1997). Consequently, changes in common silage characteristics due to
silage inoculant use cannot explain the magnitude of improvements in milk production observed.

Recent research is providing evidence, suggesting possible means by which inoculants may alter animal responses
to treated silages. Two studies indicated that lactic acid bacteria survived in rumen fluid and resulted in small but
consistent increases in pH (Weinberg et al. 2003, Weinberg et al. 2004), which should be beneficial to cell wall-
degrading microorganisms in the rumen. In addition, nine of ten inoculant LAB exhibited antimicrobial activity
when grown in broth, and the majority of extracts of silages treated with these inoculants also had antimicrobial
activity (Gollop et al. 2005). In experiments comparing 14 inoculants, none of the inoculants had a positive effect
on in vitro dry matter digestibility (IVDMD) (Filya et al. 2007). In contrast, in vitro gas production (GP) on undried
silages from those experiments was reduced in many of the inoculated silages compared to untreated control si-
lages while only minor effects on volatile fatty acid (VFA) production were observed (Muck et al. 2007). The re-
duced GP suggested that inoculated silages were producing more rumen microbial biomass yield (MBY) than un-
treated silage. Recently, Contreras-Govea et al. (2011) reported on three ensiling experiments (lucerne and two
whole-crop maizes), comparing untreated and four different microbial inoculants. In this study, there were few
differences in silage fermentation characteristics among inoculated and untreated silages, but in the in vitro rumen

Manuscript received July 2012

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fermentations, silages from two of the inoculants consistently produced more MBY than the corresponding un-
treated silages. Furthermore the increase in MBY was of an order that could explain the 3 to 5% increase in milk
production observed in cow studies (Kung and Muck 1997). These studies suggest that the effects of inoculated
silages on animal performance are due to changes in rumen microbial fermentation by an unknown mechanism.

The objective of the current experiment was to determine if extracts from silages treated with Lactobacillus plan-
tarum would affect GP, MBY and VFA production from in vitro fermentation similarly to the in vitro fermentation of
the silages. The hypothesis was that the factor in inoculated silage that enhances rumen microbial growth should
be extractable from silage.

Materials and methods

Third cut of a second year lucerne (580 g dry matter (DM) kg-1) was harvested on 31 August 2010 after field wilting
for approximately 24 h, and two maize crops were harvested, one with low DM concentration (< 300 g DM kg-1,
Maize-LDM) on 27 August 2010 and one with high DM concentration (~ 500 g DM kg-1, Maize-HDM) on 7 Septem-
ber 2010. The forages were chopped with a conventional precision-chop forage harvester and ensiled individu-
ally in 1-l glass jar mini-silos (Weck, Wher-Oftlingen, Germany) at a density of 500 g l-1 with two treatments: un-
treated control and Lactobacillus plantarum (LP, Ecosyl MTD/1, Ecosyl, North Yorkshire, UK) at 106 cfu g-1 of fresh
weight, six mini-silos per treatment. During ensiling three samples of each crop and a sample of the inoculant were
taken for enumeration of LAB by Rogosa SL agar (Muck and Dickerson 1988). Three mini-silos of each treatment
were frozen (−20 °C) after 4 and 60 d of fermentation until analysed. At opening, each mini-silo was poured into
a disinfected plastic pan and mixed to uniformity. A 20 g sample was taken, diluted 10-fold with distilled water,
and macerated for 30 s in a high-speed blender. Silage extract was filtered through 4 layers of cheesecloth, and
pH was measured immediately using a pH meter. One 20 ml aliquot sample was placed in a 50 mL polypropylene
tube, centrifuged for 20 min at 25,100 × g at 4 °C, and the supernatant decanted into a 20 ml scintillation vial and
frozen at −20 °C for later analysis of fermentation products. Fermentation products (succinate, lactate, acetate,
propionate, butyrate, and ethanol) were performed using high performance liquid chromatography (Muck and
Dickerson 1988). Two silage sub-samples of approximately 50 g each were taken for moisture analysis by freeze-
drying. The freeze-dried silage samples were ground to 1 mm and used for the determination of neutral detergent
fibre (aNDF) analysis, with heat stable amylase and sulphite, using an ANKOM fibre analyser (Ankom Technology
Corp., Fairport, NY, USA).

The remaining fresh silage from each mini-silo was chopped to a particle size of 1−4 mm using a commercial food
processor (Robot Coupe, Inc., Joliet, IL, USA) for 30 s and stored at −20 °C for later analysis of in vitro rumen fer-
mentation. The MBY and VFA production in 50 ml polypropylene plastic tubes and GP in 160 ml bottles were de-
termined on these wet-ground silages, as described previously (Contreras-Govea et al. 2011). The rumen fluid was
collected from four rumen cannulated lactating cows in the morning before feeding, following the procedure de-
scribed by Weimer et al. (2005). Donor cows were fed a TMR diet of 50:50 forage (corn silage and alfalfa silage):
concentrate. Gas production, VFA and MBY were measured after 9 and 24 h incubation at 39 °C. The MBY was cal-
culated by the difference of in vitro apparent digestibility and in vitro true DM digestibility (Blümmel et al. 1997).

In addition, 1:1 aqueous and 80% ethanol extracts of wet-ground control and inoculated silages were prepared
to study their effects on in vitro ruminal MBY and GP. Ethanol was removed from the ethanol extracts by vacuum
centrifuge, and the extract reconstituted with Type 1 water. Rumen fluid was prepared as above. Each tube or bot-
tle contained 12 ml rumen fluid, 17 ml buffer, 1 ml extract and 2 mg ml-1 glucose, which was the primary carbon
source for the rumen microorganisms. In an additional in vitro treatment, L. plantarum suspension was added in
place of extract to the buffered rumen fluid at 106 cfu ml-1.

The silage fermentation data were analysed as a 3 × 2 × 2 factorial experiment (crop × treatment × fermentation
time) and the silage extract data were analysed as a 3 × 2 × 2 × 2 factorial experiment (crop × treatment × fermen-
tation time × extract treated) using the PROC Mixed procedure of SAS (SAS Inst. Inc., Cary, NC, USA). The direct
application of L. plantarum to in vitro fluid was analysed separately from the silage extract results by a one-way
analysis of variance with crop as the fixed effect. Differences among means were tested by using the LSMEANS
statement with the PDIFF option with significance declared at p<0.05.

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Results
Silage fermentation

The inoculant was applied at slightly above the intended rate, 6.06 log
10

(cfu g-1 crop). This rate was similar to the
epiphytic population on Maize-LDM ( 6.28 log

10
[cfu g-1 crop]), but more than ten-fold higher than the LAB on the

other two crops (4.81 log
10

[cfu g-1 crop]). There were crop by treatment and crop by fermentation time interac-
tions with regard to silage characteristics (Tables 1 and 2), but no crop by treatment by fermentation time inter-
actions were found (p > 0.05), except for pH and ethanol concentration. These interactions were significant be-
cause the difference in pH between the two treatments in lucerne was much greater at day 4 (0.478) than at day
60 (0.139) whereas there were no differences in pH between treatments on a given day for either maize except
at day 4 in Maize-HDM (0.049). In the ethanol, LP-treated Maize-LDM at 4 d was similar to control (5.07 and 4.90
g kg-1 DM, respectively), but at 60 d it was greater than control (15.09 and 9.69 g kg-1 DM, respectively); in con-
trast in the other two crops, no effects of treatment were observed at day 4 or 60. The pH was lower in LP than
control in lucerne but not the two maize silages (Table 1). Inoculant treatment effects on lactic acid concentra-
tion differed by crop. In lucerne, lactic acid concentration was 45% greater in LP than control while in both maize
experiments, lactic acid was similar for both treatments (Table 1). Acetic acid concentration was different among
crops (p < 0.001) and reduced by inoculant treatment. Maize-LDM had the greatest acetic acid concentration fol-
lowed by Maize-HDM and lucerne (Table 1). Ethanol concentration in Maize-LDM was higher in LP than control
while there was no effect of treatment on ethanol in the other two crops.

Table 1. Mean silage characteristics (g kg-1 DM except as noted) by treatment across silage fermentation times.

Lucerne Maize-HDM1 Maize-LDM SEM p-value

Constituent Control LP Control LP Control LP C×T C T C×T

Dry matter
(g kg-1) 575 570 494 490 306 296 4.9 <0.001 0.13 0.81

aNDF 345 350 401 393 376 360 11.7 0.001 0.55 0.66

pH 5.30 4.99 4.19 4.18 3.79 3.77 0.008 <0.001 <0.001 <0.001

Lactic Acid 24.4 35.3 33.6 31.7 46.4 42.3 1.75 <0.001 0.25 0.001

Acetic Acid 4.63 4.38 7.28 5.95 10.80 9.14 0.429 <0.001 0.01 0.25

Ethanol 0.92 0.58 3.17 3.49 7.29 10.10 0.368 <0.001 0.01 0.01
1 HDM=high dry matter, LDM=low dry matter, LP=treated with Lactobacillus plantarum, SEM=standard error of the mean, C=crop,
T=treatment, C×T=crop×treatment interaction, aNDF=neutral detergent fibre analysed using heat-stable amylase

Silage fermentation was also affected by fermentation time (Table 2). In lucerne and Maize-LDM, the aNDF was
similar between 4 and 60 d whereas there was a decrease in aNDF with time in Maize-HDM. In all three crops, the
pH was lower at 60 d. Lactic and acetic acid concentrations were higher at 60 d for lucerne and Maize-HDM, but
similar between 4 and 60 d for Maize-LDM. Ethanol was higher in Maize-LDM at 60 d than 4 d.

Table 2. Mean silage characteristics (g kg-1 DM except as noted) by silage fermentation time across treatments.

Lucerne Maize-HDM1 Maize-LDM SEM p-value

Constituent 4 d 60 d 4 d 60 d 4 d 60 d C×D C D C×D

Dry matter
(g kg-1) 575 570 491 494 297 304 4.9 <0.001 0.67 0.52

aNDF 343 351 425 368 378 359 11.7 0.001 0.03 0.04

pH 5.66 4.63 4.36 4.00 3.82 3.74 0.008 <0.001 <0.001 <0.001

Lactic Acid 10.4 49.3 23.8 41.5 42.7 46.0 1.75 <0.001 <0.001 <0.001

Acetic Acid 1.11 7.91 4.76 8.47 9.83 10.11 0.429 <0.001 <0.001 <0.001

Ethanol 0.59 0.92 2.82 3.85 4.99 12.39 0.368 <0.001 <0.001 <0.001
1 HDM=high dry matter, LDM=low dry matter, SEM=standard error of the mean, C=crop, D=days of fermentation, C×D=crop×day interaction,
aNDF=neutral detergent fibre analysed using heat-stable amylase

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In vitro rumen fermentation

There were few effects on the 9 and 24 h in vitro rumen fermentations of the wet-ground silages relative to treat-
ment (Table 3). At 9 h with Maize-HDM, IVDMD was higher in LP than control. At 24 h in lucerne, MBY was great-
er in LP than control (p = 0.074). No other effects of treatment were observed. The highest IVDMD occurred in
Maize-HDM and lowest in lucerne, both at 9 and 24 h. At 9 h, propionate was highest in Maize-LDM and lowest in
lucerne whereas butyrate was highest in Maize-HDM and lowest in lucerne. At 24 h, all three VFA measured were
highest in Maize-HDM and lowest in lucerne. At 9 h, MBY was highest in Maize-HDM. By 24 h, MBY was highest
in lucerne and lowest in Maize-LDM. There was no effect of silage fermentation time on any of these in vitro fer-
mentation characteristics (data not shown).

Table 3. In vitro rumen fermentation profile and microbial biomass yield (MBY) of wet-ground silages at 9 h and 24 h averaged across
silage fermentation times.

Lucerne Maize-HDM1 Maize-LDM SEM p-value

Constituent Control LP Control LP Control LP C×T C T C×T

9 h

IVDMD2 9h 619 609 701 729 677 656 9.3 <0.001 0.88 0.03

Acetate
(mM)

49.8 50.6 49.4 48.9 49.0 47.3 1.25 0.29 0.65 0.61

Propionate
(mM)

13.5 13.7 16.4 16.6 18.3 17.6 0.62 <0.001 0.87 0.68

Butyrate
(mM)

6.7 6.6 12.1 12.2 10.1 10.3 0.31 <0.001 0.86 0.95

MBY
(mg/g DM)

329 325 418 429 315 268 17.4 <0.001 0.36 0.24

24 h

IVDMD 24h 645 661 846 836 768 760 11.5 <0.001 0.94 0.47

Acetate
(mM)

53.8 52.7 73.0 71.8 60.9 62.2 1.18 <0.001 0.72 0.50

Propionate
(mM)

14.6 15.0 26.2 26.5 20.7 21.2 0.71 <0.001 0.52 1.00

Butyrate
(mM)

8.0 8.6 20.6 21.0 13.7 13.9 0.55 <0.001 0.35 0.93

MBY
(mg/g DM)

314 347 241 241 196 193 8.3 <0.001 0.15 0.07

1 HDM=high dry matter, LDM=low dry matter, LP=treated with Lactobacillus plantarum, C=crop; T=treatment, C×T=crop×treatment
interaction, SEM=standard error of the mean
2 IVDMD=In vitro dry matter digestibility, MBY=Microbial biomass yield estimated by the difference of in vitro true digestibility and in vitro
apparent digestibility (Blümmel et al. 1997).

In vitro rumen fermentation of the water and ethanol silage extracts had an effect on MBY and GP, but the effect
was different among crops and between treatments (Table 4). The MBY of water extracts was greater in control
than LP in lucerne, while no effect of treatment was observed in either maize. In contrast, the MBY of ethanol ex-
tracts was unaffected by treatment, but there were trends for greater MBY in LP than control in lucerne and the
opposite in the two maizes. Averaging across crops and treatments, the MBY was 4.7% greater in the ethanol ex-
tract than the water extract (Table 4). The GP in the water extracts was not affected by treatment (Table 4). In the
ethanol extracts, GP was higher in LP than control in lucerne and Maize-LDM.

Effects of treatment on the VFA profiles of the in vitro rumen fermentations of silage extracts were observed in
the lucerne extracts (Table 5). The acetate concentrations from ethanol extracts of the LP treatment were higher
than the LP water extract and both control extracts. Propionate concentrations were reduced in the ethanol ex-
tracts compared to the water extracts. Butyrate concentration was lower in the ethanol extracts of the control
compared to the other extracts. There was no effect of silage fermentation time on VFA concentrations. However,
there were significant interactions with treatment (data not shown); acetate and butyrate concentrations were
lower in the control extracts at 4 d than at 60 d and than in the LP extracts at 4 and 60 d, which were all similar.

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Table 4. Microbial biomass (MBY, mg g-1 DM) and gas production (GP, mL g-1 DM) from water or ethanol silage extracts and glucose
after 24 h in vitro rumen fermentation.

Lucerne Maize-HDM1 Maize-LDM SEM p-value

Extract Control LP Control LP Control LP C×T C T C×T

MBY

Water
extract

20.7 15.7 15.9 16.5 16.6 15.9 1.32 0.21 0.20 0.02

Ethanol
extract

16.4 18.8 19.9 18.1 17.1 15.7 1.32 0.21 0.20 0.02

LP-DA2 13.5 13.3 12.0 1.31 0.70

Water
extract

35.2 36.0 19.8 21.3 25.4 27.2 1.06 <0.001 0.02 0.08

Ethanol
extract

33.7 37.3 21.9 19.7 24.5 28.2 1.06 <0.001 0.02 0.08

LP-DA 24.7 16.8 21.6 1.49 0.01
1 HDM=high dry matter, LDM=low dry matter, LP=treated with Lactobacillus plantarum, C=crop, T=treatment, C×T=crop×treatment
interaction, SEM=standard error of the mean
2 LP-DA= direct addition of LP to rumen inoculum, no silage extract.

Table 5. Volatile fatty acid concentrations (mM) from water or ethanol lucerne silage extracts and glucose after 24 h in vitro rumen
fermentation.

Control LP1

Water Ethanol Water Ethanol SEM p-value

Acetate 37.2 37.9 36.1 41.4 0.38 0.001

Propionate 11.3 9.3 11.0 10.1 0.17 0.005

Butyrate 7.2 6.6 7.0 7.1 0.10 0.004
1 LP=treated with Lactobacillus plantarum, SEM=standard error of the mean

The direct application of LP to the in vitro rumen inoculum produced less MBY and GP than the silage extracts
(Table 4). Extracts for each crop were performed in separate in vitro runs and tubes with LP added directly to ru-
men fluid were included in each run. So, similar MBY and GP values for direct application of LP would be expect-
ed across crops. That was true for MBY but not GP, where GP was lower in the Maize-HDM in vitro runs than in
the other two crops.

Discussion

A probiotic effect of silage microbial inoculants on animal performance was proposed by Weinberg and Muck
(1996). Even though the mechanism by which silage microbial inoculants enhance animal performance has not
been elucidated, better preservation of nutrients in the silage could explain in part the animal performance effect
(Muck et al. 2007, Contreras-Govea et al. 2011). In the current study, the hypothesis was that a factor in inocu-
lated silage that enhances rumen microbial activity should be extractable from silage. Therefore, three different
crops harvested separately were inoculated with a specific strain of LP that has usually shown a positive effect on
silage fermentation and animal performance (Kung et al. 2003).

Silage fermentation characteristics of the three crops with or without LP were typical of silages ensiled at high and
more typical DM concentrations. Because of their high DM concentrations, the pH and fermentation products of
lucerne and Maize-HighDM silages were different between day 4 and day 60, indicating that active fermentation
by LAB was not complete after 4 d (Table 2). In contrast, there were no differences in silage fermentation charac-
teristics in Maize-LDM between the two days with the exception of an increase in ethanol. Given that lactic acid
did not increase between days 4 and 60, the increase in ethanol in the Maize-LDM silages may have been due to
the activity of yeasts (McDonald et al. 1991).

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The LP treatment affected silage fermentation, compared to control, in lucerne but not appreciably in the two
maize experiments. In lucerne, LP decreased pH and increased lactic acid compared to control (Table 1), and the
effect was evident at both days 4 and 60 (data not shown). This is what would be expected when an inoculant like
L. plantarum is used. For example, Filya et al. (2007) also reported lower pH and higher lactic acid in the inoculated
lucerne silage compared with control when ensiled at high DM concentrations. In contrast, the only effect of LP on
silage fermentation in Maize-HDM was a lower pH at day 4 (4.339 vs. 4.388), and in Maize-LDM a higher ethanol
concentration at day 60 (15 vs. 10 g kg-1 DM). The lack of an inoculant effect is not uncommon. Muck and Kung
(1997) in a survey of published studies reported that 60% of the time silage microbial inoculants decreased pH
compared to uninoculated treatments. So, there are a substantial numbers of instances where an inoculant has not
affected silage fermentation. In the case of Maize-LDM, the epiphytic LAB population was similar to the inoculant
application rate and may have competed effectively with the inoculant. In Maize-HDM, the epiphytic LAB popula-
tion was lower than the inoculant application rate. Perhaps other epiphytic bacteria such as enterobacteria domi-
nated the early fermentation (Pahlow et al. 2003). Unfortunately the only microbial group analysed was the LAB.

The absence of a consistent effect of LP across crops on the in vitro rumen fermentation at 9 h and 24 h (Table 3)
was not unexpected. Muck and Kung (1997) reported that a positive effect of inoculant treatment on in vitro true
DM digestibility occurred in only 30% of the studies reviewed. In addition, Muck et al. (2007) reported no consist-
ent effect of microbial inoculants on VFA composition of in vitro rumen fermentations of inoculated and untreated
lucerne silages. The MBY at 9 h and 24 h in the current study did not show a consistent effect of LP across crops in
contrast to our earlier study (Contreras-Govea et al. 2011). In Contreras-Govea et al. (2011), MBY was greater in the
LP-treated silages than control across lucerne, maize and brown-midrib maize, but this effect was not consistent
across the other inoculants tested. In our study, the MBY at 9 h was unaffected by inoculant treatment in any of
the crops (Table 3). At 24 h, MBY was higher in LP-treated lucerne silages than control silages (p = 0.074) whereas
MBY was unaffected by treatment in the maize silages. Because the inoculant only affected the ensiling of the lu-
cerne, the lack of treatment effects in the in vitro fermentations, particularly in the maize silages, is not surprising.

The hypothesis of this study was that if the mechanism that improves animal performance is produced by the mi-
crobial inoculant during fermentation, it should be extractable from the silage. The 80% ethanol extract was ex-
pected to remove more complex carbohydrates and N compounds than the water extract. Averaging across crops
and treatments, MBY was 4.7% greater in the ethanol silage extract than water silage extract (Table 4). MBY from
the extracts in maize were unaffected by treatment, but given the few effects of treatment in silage fermentation
and in vitro fermentation of these silages, the lack of effect was expected. In the lucerne, the water extract of
the control produced a higher MBY than the water extract of the LP silage and the ethanol extract of the control.
These results suggest that the factor causing higher MBY in the LP-treated lucerne silage at 24 h in vitro fermen-
tation is not water-soluble. While there was only a numerical trend for MBY to be higher in the ethanol extract of
LP-treated lucerne silages compared to control, the ethanol extracts from the LP-treated lucerne silages produced
more GP, acetate, propionate and butyrate than the control ethanol extracts (Tables 4 and 5). Such results stand
in contrast to the in vitro rumen fermentations of the silages in this study and earlier ones (Muck et al. 2007, Con-
treras-Govea et al. 2011) where LP-treatment had no effect on or reduced GP and had little or no effect on VFA
production. Consequently these results suggest that the substances causing increased MBY in in vitro fermenta-
tions of LP-treated silages were in neither the water nor ethanol extract.

Finally, MBY and GP from direct addition of LP to in vitro rumen fluid were lower than silage extracts (Table 4).
These results suggest that the inoculant LAB are not having any direct effect on in vitro rumen fermentation or at
least that the extracts are providing more fermentable substances than the bacteria themselves.

Conclusion

In this study, Lactobacillus plantarum MTD/1 appeared to dominate in only one of the three crops ensiled, a lu-
cerne. In that crop, in vitro rumen fermentation of the silages produced more MBY at 24 h in the LP-treated silag-
es, consistent with earlier studies. In vitro fermentation of the water extracts of the lucerne silages produced no
significant treatment differences in fermentation products with the exception of reduced MBY in the LP-treated
extracts, indicating the factor affecting in vitro fermentation of the inoculated silages was not in the water extract.
The ethanol extracts produced in vitro results that were also not consistent with the in vitro results of the silages.
Consequently it appears that the factor in in vitro fermentation of inoculated silages causing increased MBY was
in neither the water or ethanol extracts. However, there is the need for further research to confirm these results.

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Acknowledgements

The authors wish express their appreciation for the technical assistance of U.C. Hymes-Fecht and J.A. Boyd.

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Silage extracts used to study the mode of action of silageinoculants in ruminants
Introduction
Materials and methods
Results
Silage fermentation
In vitro rumen fermentation

Discussion
Conclusion
Acknowledgements
References