RESEARCH PAPER

https://doi.org/10.1071/AN20274

Lemongrass essential oil reduces Mombasa grass silage gas

losses, fibre content and pH after aerobic exposure
Thainá M. Garcia A, Estéfani Capucho A, Roberto Cantoia Júnior B, Mauricio F. B. Burró A, Rebeca R. Noernberg A,
Elissandra M. C. Zilio C, Mariana Campana A, Tiago A. Del Valle D,* and Jozivaldo P. G. de Morais A

For full list of author affiliations and
declarations see end of paper
*Correspondence to:
Tiago A. Del Valle
Departamento of Animal Sciences, Rural
Sciences Center, Federal University of Santa
Maria, Santa Maria 97105-900, Brazil
Email: tiago.valle@ufsm.br
Handling Editor:
Di Mayberry
ABSTRACT
Context. Ensiling is commonly used to conserve tropical grasses, but low water-soluble carbohy-
drates content and high moisture content in the grass impair silage fermentation characteristics.
Essential oils may be used as silage additives to decrease fermentative losses and improve the
nutritional value of silage, and aerobic stability, and in vitro DM disappearance. Aims. The present
study aimed to evaluate the effect of including lemongrass essential oil (LEO) on the fermentative
characteristics, fermentative losses, chemical composition, in vitro disappearance and aerobic stability
of Mombasa grass (Megathyrsus maximum) silage. Methods. Mombasa grass was ensiled for 60 days in
32 experimental silos (15-L plastic buckets, 28 cm diameter and 25 cm high), in a randomly blocked
design. LEO was included at 0.00, 0.67, 1.33 and 2.00 mL per kg of silage fresh matter. Silos were
weighed every 15 days, pH and temperature were measured immediately after the silos were
opened, and subsamples of silage were taken to measure the fermentative profile, composition of
silage effluent, chemical composition and in vitro degradation of silage. Key results. Increasing the
amount of LEO in Mombasa grass silage caused a linear reduction in ammonia–nitrogen, ethanol
(P < 0.05), acid detergent fibre of silage, gas losses and silage pH after aerobic exposure. There
was no impact (P > 0.05) on organic acid concentration, effluent production or DM recovery
(P > 0.05). Conclusions (Tilley and Terry 1963). Adding LEO to Mombasa grass silage at up
to 2.00 mL per kg fresh material (almost 7.5 mL/kg DM) reduces gas losses, ammonia–nitrogen
and acid-detergent fibre concentration, and silage pH after aerobic exposure. However, it is not
sufficient to improve DM recovery and in vitro disappearance of silage. Implications. Lemongrass
essential oil shows a positive effect on Mombasa grass silage fermentation, fibre content and silage
parameters after aerobic exposure, providing a useful additive in this silage.

Keywords: ammonia, chemical composition, digestibility, fatty acids, feed quality, forage, perennial
grasses, silage.

Introduction

Megathyrsus maximum (c.v. Mombasa) is a high-yielding tropical grass (4.8 ton DM/ha/
cycle; Müller et al. 2002), with production sometimes exceeding grazing animals’
Received: 2 June 2020
requirements. In cases where grass production exceeds the target height for grazing,
Accepted: 30 May 2022 grass can be cut and ensiled to ensure that forage is not wasted (Daniel et al. 2019).
Published: 8 July 2022 However, low water-soluble carbohydrates content and high moisture content impair
grass silage fermentation (Evangelista et al. 2004), increasing fermentative losses due to
Cite this: high pH, ammonia–nitrogen (NH3-N) and butyric acid (McDonald et al. 1991). To
Garcia TM et al. (2022) reduce losses and improve the nutritional value of silage, several additives have been
Animal Production Science
studied to manipulate the fermentative characteristics of grass silage (Avila et al. 2009;
doi:10.1071/AN20274
Oliveira et al. 2014).
Essential oils are blends of secondary metabolites obtained from the plant volatile
© 2022 The Author(s) (or their
employer(s)). Published by
fraction by distillation (Gershenzon and Croteau 1993). Essential oils show anti-oxidant
CSIRO Publishing. activity (Amorati et al. 2013) and have antimicrobial properties, which may impact
ruminal fermentation (Calsamiglia et al. 2007). Recent studies have shown that adding
T. M. Garcia et al.
essential oils inhibits deamination during ryegrass ensiling
(thymol, eugenol and cinnamaldehyde; Foskolos et al.
2016) and decreases growth of yeast in barley silage
(eugenol, carvacrol and thymol; Chaves et al. 2012).
Lemongrass (Cymbopogon citratus) essential oil (LEO)
contains flavonoids, phenolic compounds and terpenoids,
such as citral α, citral β, nerolgeraniol, citronellal, terpinolene
and geranyl methylheptenone. It shows antibacterial,
antifungal (Valero and Salmerón 2003) and anti-oxidant
effects (Cheel et al. 2005). Cantoia et al. (2020) evaluated
the impact of including LEO in sugarcane silage, and
reported that intermediary doses (between 1.53 and
2.22 mL/kg) reduced ethanol concentration and fermentative
losses, and increased nutritional value and in vitro DM
disappearance.
The responses of silage fermentation and feed degradation
to the addition of essential oils likely depend on the type of
forage being ensiled and the dose of essential oil. We
hypothesised that increased doses of LEO could inhibit
secondary fermentation, and reduce pH and fermentative
losses, thus increasing in vitro DM disappearance, aerobic
stability and, consequently, the nutritional value of grass
silage. This study aimed to assess the effect of increasing LEO
doses in Mombasa grass silage on fermentative character-
istics, gas and effluent production, chemical composition,
in vitro DM, neutral detergent fibre (NDF) disappearance
and aerobic stability.
Materials and methods
The experiment was performed in the Group of Agriculture
Study and Labour of Agricultural Sciences Centre, Federal
University of Sao Carlos, in Araras, São Paulo State, Brazil,
between December 2018 and February 2019. Experimental
procedures were performed under the Ethics Committee’s
approval from the Federal University of São Carlos
(#1395120219).
Experimental design and silage preparation
The experiment compared the fermentation characteristics of
Mombasa grass silage with four different levels of LEO: 0.00
(control), 0.67, 1.33 and 2.00 mL of LEO per kg of grass silage
(as-is basis). The silage was prepared in 32 experimental silos
in a blocked randomised design, with eight replicates per dose
level. Doses were based on the study by Cantoia et al. (2020),
which reported improved fermentative characteristics,
chemical composition and DM disappearance using doses
between 1.53 and 2.22 mL/kg of fresh matter. The grass
was manually ensiled for 60 days in 15-L silos comprising a
plastic bucket (PVC tubes) with 28-cm diameter, 25-cm
high and equipped with a Bunsen valve to allow gas to escape.
Mombasa grass was manually harvested from four
different fields at 5-cm height and chopped in a stationary
B
Animal Production Science
hammer mill (TRF300®; Trapp, Jaguará do Sul, Santa
Catarina State, Brazil). Mombasa grass for each silo was
individually weighed and manually mixed with LEO
(ANVISA registration 25351.179913/2017–23) purchased
from Quinarí (Ponta Grossa, Paraná State, Brazil). Five
kilograms of sand were placed at the bottom of silos with a
nylon screen to separate the chopped grass and quantify
effluent production. The unloaded silo (containing sand and
nylon screen) was weighed, the material was compacted
(650 kg/m3) and the silos were sealed.
Sampling and analyses
Mombasa grass height was analysed before harvest using a
2-m scale. Unchopped grass samples were obtained and dried
in a forced-air oven (60°C for 72 h) to evaluate morphological
composition. The morphological composition was evaluated
by manual separation of leaf lamina, stem, inflorescence
and dead material. Chopped grass samples were collected,
dried in a forced-air oven (60°C for 72 h), and used for
particle size evaluation (Maulfair et al. 2011) and chemical
analysis.
The total weight of each silo was recorded every 15 days
after ensiling and at the opening using a 5-g sensitive scale
(Mettler Toledo, Barueri, São Paulo State, Brazil). After
60 days, silos were opened, and the top and bottom 5 cm of
silage was removed and discarded. Silage of the centre of
silos was mixed and a sample of 3.5 kg was manually
collected. This sample was split into three subsamples for
measurement of (1) pH, NH3-N and acid concentration in
silage fluid (200 g), (2) chemical analysis and in vitro
disappearance of DM and NDF (300 g), and (3) aerobic
stability of silage (3 kg). After all silage removal, silos were
weighed to quantify effluent production.
The first 200-g subsample was pressed in a hydraulic press
(PHE-45®; Engehidro, Piracicaba, São Paulo State, Brazil) to
obtain silage fluid. The silage fluid was squeezed through
cheesecloth layers, and pH was measured using a pH digital
potentiometer (LUCA-210®; Lucadema, Sao José do Rio
Preto, São Paulo State, Brazil). Subsamples of the silage
fluid were then taken for analysis of NH3-N, ethanol, and
lactic, acetic, propionic and butyric acids.
NH3-N was analysed using the Kjeldahl method (method
984.13; AOAC 2000), without pre-digestion. Lactic acid
concentration was measured using the spectrophotometric
method of Pryce (1969). Briefly, samples were diluted in
phosphoric acid and centrifuged at 3000g for 5 min, and
the supernatant was homogenised with sulfuric acid and
heated to 75°C for 2.5 min. After cooling, a colour reagent
(4-phenyl phenol; Sigma Aldrich, St. Louis, MO, USA) was
added. The sample was then heated to 90°C for 1.5 min,
and analysed after cooling using a spectrophotometer at
560 nm. For analysis of ethanol, acetic, propionic, butyric,
valeric, iso-valeric and isobutyric acids, silage fluid was
mixed with formic acid at a ratio of 4:1. A gas
www.publish.csiro.au/an Animal Production Science

chromatograph (GC-2010 Plus chromatograph; Shimadzu, 25 g of silage (fresh matter) was collected from the middle
Barueri, Brazil) equipped with an AOC-20i auto-sampler, of the bucket, diluted in 100 mL of distilled water and pH
Stabilwax-DA™ capillary column (30 m, 0.25 mm internal evaluated as previously described, after 15 min of standing
diameter, 0.25 μm df; Restek©), and a flame ionisation time (Bernardes et al. 2019).
detector was used to determine organic acids and ethanol,
as described by Del Valle et al. (2019). A 1-μL sample was Calculations and statistical analyses
injected with a split ratio of 40:1, using helium as the
Non-fibre carbohydrate (NFC) was calculated according to
carrier gas at 42 cm/s, in a chromatographic run of
Hall (2000):
11.5 min. The temperatures of the injector and detector


were 250 and 300°C, respectively. Column temperature g
started with a gradient from 40 to 120°C at 40°C/min rate, NFC = 1000 − ðNDF + CP + ash + EEÞ
kg DM
followed by heating at 10 and 120°C/min rate between 120
and 180°C, and from 180 to 240°C, respectively. Method Gas losses (GL), effluent production (EP) and DM recovery
calibration was made using diluted solutions of the WSFA-2 were estimated using the equations of Jobim et al. (2007). GL
standard (Ref. 47056; Supelco©) and ethanol (Ref. 459828; were calculated as the difference between whole silo weight
Sigma-Aldrich©). Peak detection was performed using the at ensiling (WSWE) and whole silo weight at the opening
GC solution version 2.42.00 software (Shimadzu©). The (SWO), as a fraction of ensiled DM (EDM):
sum of valeric, butyric and isobutyric acids were reported

as branched-chain fatty acids. g WSWE ðgÞ − WSWO ðgÞ
GL =
The second subsample of silage (300 g) was frozen (−20°C) kg DM EDM ðkgÞ
until subsequent chemical analyses and in vitro assay. After
defrosting, the sample was dried at 60°C in a forced-air where EDM was calculated as:
oven for 72 h and ground in a knife mill (SL-31; Solab

g DM
Científica, Piracicaba, Brazil) through a 2-mm sieve. Samples EDM = ðWSWE − ESWEÞ × DM content
g fresh matter
(almost 150 g) were then reground in a knife mill using a
1-mm sieve, and analysed for DM (method 950.15; AOAC Effluent production was calculated based on the difference
2000), ash (method 942.05; AOAC 2000), crude protein between silo weight at the empty silo weight at opening
(CP; N × 6.25; Kjeldahl method 984.13; AOAC 2000), acid (ESWO) and empty silo weight at ensiling (ESWE) as a
detergent fibre (ADF; method 973.18; AOAC 2000), NDF fraction of ensiled DM:
without the addition of α-amylase and sodium sulfite,

expressed including residual ash, according to Van Soest g ½ESWO ðgÞ − ESWE ðgÞ
EP =
et al. (1991). kg DM EDM ðkgÞ
Disappearance of DM and NDF were determined in vitro,
according to the method of Tilley and Terry (1963), as DM recovery was calculated by dividing DM at opening
modified by Holden (1999). Samples (processed through a (DMO) by the EDM, as follows:
2-mm sieve) were placed in bags of non-woven fabric tissue
(5 × 5 cm and 100 DM/m2; Casali et al. 2008) and DMO ðkgÞ
DMR =
incubated for 48 h at 38°C in an in vitro incubator EDM ðkgÞ
(NL162®; New Lab, Piracicaba, Brazil). Three blank and
Data were analysed using PROC MIXED of SAS (version
three control (standard samples) bags were included in
9.4, SAS Institute., Cary, NC, USA), according to the
each run. The inoculum was a mix of McDougall (1948)
following model:
buffer and ruminal fluid obtained from two dairy heifers
maintained in Mombasa grass pasture, without concentrate Y ijk = μ + LEOi + bj + eijk
supplementation. After incubation, samples were washed in
running tap water, pre-dried at 60°C for 24 h and dried for with bj ≈ Nð0, σ 2b Þ and eijk ≈ Nð0, σ 2e Þ, where Yijk is the
1 h at 105°C (Detmann et al. 2012) to obtain undigestible dependent variable; μ is the overall mean; LEOi is the fixed
DM. Samples were then analysed for NDF content, as effect of lemongrass essential oil (i = 1–4); bj σ 2b and σ 2e are
previously described, to obtain undigestible NDF. DM and variances associated with blocks and residual random
NDF in vitro disappearance was corrected by blanks. effects, respectively. Doses of LEO were tested for linear
The final subsample (3 kg) was placed in a plastic and quadratic effects using polynomial regression.
bucket and maintained in a controlled temperature room Silage pH and temperature after aerobic exposure were
(21.6 ± 1.73; mean ± s.d.) for 5 days. The temperature of analysed as repeated measures, according to the following
the silage mass centre was recorded every 8 h using spit model:
thermometers (K29–5030®; Kasvi Produtos Laboratoriais,
Pinhais, Paraná State, Brazil). Every 24 h, approximately Y ijkl = μ + LEOi + bj + ωijk + T l + LEO × T il + eijkl

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T. M. Garcia et al. Animal Production Science

with bj ≈ Nð0, σ 2b Þ; ωijk ≈ Nð0, σ 2ω Þ and eijkl ≈ MVN(0,R), where
Yijkl is the dependent variable; μ is the overall mean; LEOi is
the fixed effect of lemongrass essential oil (i = 1–4), bj σ 2b and
σ 2ω are variances associated with blocks and silos random
effects, respectively; MVN stands for multivariance analysis
with normal distribution; and R is a matrix of variance and
covariance due to repeated measures. The Bayesian method
was used to choose FA and CSH as the best matrixes for
silage pH and temperature, respectively. For all analyses,
P ≤ 0.05 was considered effect, and 0.05 < P ≤ 0.10 was
defined as a tendency.
Results
Composition of Mombasa grass prior to ensiling was
268 g DM/kg, 807 g NDF/kg DM and 47.0 g CP/kg DM
(Table 1). The average in vitro disappearance of DM and NDF
of non-ensiled Mombasa grass was 527 and 474 g/kg DM,
respectively. This low nutritional value is associated with
the high height during the harvest (160 ± 16.8 cm), which
resulted in 477 g/kg DM of stalk.
Adding LEO to Mombasa grass silage did not affect
(P ≥ 0.16) silage pH or the concentrations of lactic, acetic,
butyric and branched-chains fatty acids (Table 2). Doses of
LEO linearly reduced (P ≤ 0.04) NH3-N and ethanol
concentration, and quadratically affected propionate
concentration (P = 0.02). Intermediary doses of LEO (0.67
and 1.33 mL/kg) increased the concentration of propionate
in relation to control and the highest doses (2.00) of LEO.
LEO had no effect on effluent production (P ≥ 0.33) and
linearly reduced gas losses (P = 0.02; Table 3). Effluent
production averaged 51.3 g/kg as-fed (191 g/kg DM) and
was responsible for 83% of total losses, thus, LEO did not
affect total fermentative losses and DM recovery of grass
silage. LEO did not affect the DM, organic matter, NDF,
non-fibre carbohydrate, ether extract or CP content of
Mombasa grass silage (P ≥ 0.51), or disappearance of DM
or NDF (P ≥ 0.30; Table 4). LEO linearly decreased ADF
concentration (P = 0.02).
Inclusion of LEO tended to affect silage pH and
temperature after aerobic exposure (P ≤ 0.08; Figs 1 and 2).
Average temperatures up to 120 h were 1.19, 0.94, 1.22 and
0.83°C above ambient temperature for silages treated with
0.00, 0.67, 1.33 and 2.00 mL/kg of LEO, respectively.
Neither the linear nor quadratic effect of LEO doses were
observed (P ≥ 0.15) on temperature after aerobic exposure,
but LEO linearly reduced silage pH after aerobic exposure
(P = 0.03), regardless of the time of evaluation (P = 0.93).
Discussion

Table 1. Chemical composition, particle size and morphological We hypothesised that increased LEO doses could inhibit
composition of Mombasa grass prior to ensiling. secondary fermentation of Mombasa grass silage, reducing
silage pH and fermentative losses, improving in vitro DM
Item Mean s.d.
disappearance, aerobic stability and nutritional value of grass
Chemical composition (g/kg DM) silage. Inhibition of secondary fermentation was evidenced by
DM (g/kg as-fed) 268 13.3 the reduction of NH3-N and ethanol, and there was also a
Organic matter 989.5 1.64 reduction in gas fermentative losses. Inclusion of LEO also
Neutral detergent fibre 807 16.0 decreased silage pH after aerobic exposure. However, this
Acid detergent fibre 511 78.5
was not accompanied by a reduction in silage pH, increased
in vitro DM disappearance or changes in the nutritional value.
Crude protein 47.0 2.49
In the present study, LEO addition in grass silage linearly
Non-fibre carbohydrate + ether extract 136 19.1
reduced NH3-N and ethanol concentration. Evaluating oil
In vitro disappearance (g/kg) extracted from microalgae addition in sorghum ensiling,
DM 527 27.3 Chen et al. (2018) reported increased acetic acid and
Neutral detergent fibre 474 42.7 ethanol in microalgae-treated silos, and speculated that
Particle size (g/kg as-fed) microalgae fatty acids induced a heterolactic fermentation.
>19 mm 751 77.8
In our research, none of these effects were observed.
Excessive NH3-N production is toxic to acidogenic bacteria
8–19 mm 194 68.8
during fermentation, because the unionised NH3-N can
4–8 mm 10.7 6.20 cross cell membranes by passive diffusion, resulting in
<4 mm 44.1 11.8 proton imbalance (Ward et al. 2014). As grass silage usually
Morphological composition (g/kg as-fed) has low substrate to lactic acid bacteria fermentation, a
Stalk 477 244 reduced NH3-N production favours silage fermentation.
Leaf 417 205 Evaluating increasing doses (up to 2.00 g/kg as fed) of
thymol, eugenol, carvacrol and cinnamaldehyde essential
Dead 106 190
oil in ryegrass silage, Foskolos et al. (2016) observed
Height (cm) 160 16.8
decreased NH3-N concentration. Like those authors, we

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Table 2. Fermentation characteristics of Mombasa grass silage containing different doses of lemongrass essential oil upon opening the silos.
Item Level of LEO (mL/kg grass silage) s.e.m.A PB
0.00 0.67 1.33 2.00 Treat. Linear Quad.
pH 4.84 4.85 4.83 4.76 0.075 0.72 0.34 0.53
NH3-N (g/kg N) 27.8 24.9 25.5 21.0 1.47 0.10 0.04 0.69
Ethanol (g/kg DM) 4.43 3.67 2.89 2.87 0.744 0.04 0.01 0.38
Organic acids (g/kg DM)
Acetic 22.9 26.2 22.8 17.9 1.30 0.16 0.11 0.11
Lactic 24.4 14.6 16.5 28.5 5.80 0.57 0.70 0.18
Butyric 9.47 11.1 11.3 9.68 3.111 0.61 0.88 0.19
Propionic 1.10 1.26 1.39 1.13 0.270 0.07 0.53 0.02
C
BCFA 0.696 0.815 0.744 0.608 0.1003 0.17 0.26 0.06
A
Standard error of the mean.
B
Probabilities: Treat., treatment (lemongrass oil doses) effect; Linear, linear effect of lemongrass oil dose; Quad., quadratic effect of lemongrass oil dose; Dev., deviation
of quadratic (cubic) effect of lemongrass oil dose.
C
Branched-chain fatty acids.

Table 3. Fermentative losses and DM recovery of Mombasa grass silage containing different doses of lemongrass essential oil.
Item Level of LEO (mL/kg grass silage) s.e.m.A PB
0.00 0.67 1.33 2.00 Treat. Linear Quad.
Fermentative losses (g/kg as-fed)
Effluent 54.6 46.2 56.8 47.7 8.73 0.33 0.63 0.93
Gas 11.5 10.9 10.6 9.55 1.88 0.10 0.02 0.69
Total 66.0 57.1 67.3 57.2 9.34 0.32 0.47 0.90
Fermentative losses (g/kg DM)
Effluent 203 172 212 178 31.6 0.34 0.66 0.95
Gas 41.7 39.5 38.5 34.6 6.53 0.10 0.02 0.69
Total 245 211 250 213 33.5 0.33 0.50 0.92
DM recovery 856 823 821 883 14.6 0.52 0.39 0.25
A
Standard error of the mean.
B
Probabilities: Treat., treatment (lemongrass oil dose) effect; Linear, linear effect of lemongrass oil dose; Quad., quadratic effect of lemongrass oil dose.

agree that NH3-N reduction occurred due to inhibition of
deamination with essential oils addition during the ensiling.
The reduction of ethanol concentration in LEO-treated silos
was previously observed by Cantoia et al. (2020). In their
study, increasing LEO doses in sugarcane ensiling reduced
ethanol concentration, which was associated with reducing
mould and yeast growth, and ethanol production due to the
antifungal effect of LEO (Shah et al. 2011). Although LEO
potentially inhibited NH3-N and ethanol production, no
effects were observed on concentrations of lactate, acetate,
butyrate and branched-chain fatty acids. It was hypothesised
that those effects on NH3-N and ethanol could be associated
with increased organic acid concentration and reduced silage
pH. The addition of LEO quadratically affected propionate
concentration; intermediary doses of LEO increased silage
propionate content. This effect could occur due to inhibited
heterolactic fermentation. However, the difference among
values is insufficient to presume other effects on silage
losses and chemical composition.
Among the essential oils studied by Foskolos et al. (2016),
eugenol and cinnamaldehyde showed no impact on lactic acid
and silage pH. In contrast, thymol and carvacrol reduced lactic
acid. and increased silage pH. The lack of LEO effect on lactic
acid concentration and silage pH reinforces that this essential
oil has minimal effects on lactic acid bacteria metabolism.
Cantoia et al. (2020), in contrast, reported a negative effect
of LEO on sugarcane lactic acid concentration and increased
silage pH, but it is necessary to highlight that the biochemical
and microbiological environment of sugarcane silage are
entirely different from those of grass silage.
The inclusion of LEO linearly reduced gas losses, which
seems to be associated with decreased ethanol production.
Silages with higher ethanol concentration show improved
gas losses, especially when samples are oven dried.

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Table 4. Chemical composition and in vitro disappearance of Mombasa grass silage containing different doses of lemongrass essential oil.
Item Level of LEO (mL/kg grass silage) s.e.m.A PB
0.00 0.67 1.33 2.00 Treat. Linear Quad.
Chemical composition (g/kg DM)
DM (g/kg as-fed) 246 235 244 251 4.4 0.51 0.47 0.25
Organic matter 988.1 988.4 988.3 988.5 0.48 0.90 0.55 0.86
Neutral detergent fibre 755 775 754 737 15.3 0.73 0.49 0.43
Acid detergent fibre 520 531 485 485 7.8 0.05 0.02 0.69
Crude protein 44.0 43.9 42.6 44.5 1.68 0.87 0.97 0.57
Non-fibre carbohydrate + ether extract 173 166 191 213 15.0 0.52 0.19 0.55
In vitro disappearance (g/kg)
DM 503 501 497 519 4.2 0.30 0.27 0.17
Neutral detergent fibre 418 446 426 434 7.7 0.50 0.62 0.46
A
Standard error of the mean.
B
Probabilities: Treat., treatment (lemongrass oil dose) effect; Linear, linear effect of lemongrass oil dose; Quad., quadratic effect of lemongrass oil dose.

3.0 Probabilities (P):

Temperature (°C difference from ambient temperature)

Treat.: 0.08
2.5 Time <0.01
Treat. × time: 0.76
Linear: 0.15
2.0 Quadratic: 0.56

1.5

1.0

0.5

0.0
0 10 20 30 40 50 60 70 80 90 100 110 120
Time (hours after aerobic exposure)
−0.5

−1.0

Fig. 1. Temperature after aerobic exposure of Mombasa grass silage treated with increasing doses of lemongrass
essential oil. Lemongrass essential oil doses: 0.00 (– ▪ –), 0.66 (– □ –), 1.33 (– ♦ –) and 2.00 (– ⋄ –) mL/kg.

However, silage showed significant effluent production, digestible fraction of fresh material. However, effluent
which averaged 83% of total fermentative losses and production carries water-soluble constituents of feed,
became null the effect of LEO on total fermentative losses comprising chemical composition and nutritional value. In
and DM recovery. Other studies (Rigueira et al. 2013; Viana the present study, LEO linearly reduced ADF, but no effect
et al. 2013) have already highlighted grass DM content as was observed on silage chemical composition, which seems
the primary determinant of effluent production. Although a direct effect of reduced gas losses in LEO-treated silages,
general effluent production observed in the present study and no effect of LEO on total fermentative losses and DM
had been lower than that reported by those authors, LEO recovery, respectively. Other studies (Cochran et al. 1986;
has no adsorbent effect and no effect was observed on Andrae et al. 2001) negatively associated ADF content with
effluent production. DM and NDF disappearance, which was not observed in
Increased gas losses have been associated with the lower the current study. In essence, the higher level of NDF,
nutritional value of silages (Pedroso et al. 2005), because compared with ADF, decreased accuracy, as observed in
lactic acid bacteria fermentation consumes the most s.e.m. (7.8 g/kg for ADF and 15.3 g/kg for NDF) in the

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Probabilities (P): Andrae JG, Hunt CW, Pritchard GT, Kennington LR, Harrison JH, Kezar W,
Treat.: 0.07
Time <0.01
Mahanna W (2001) Effect of hybrid, maturity, and mechanical
5.3 Treat. × time: 0.93 processing of corn silage on intake and digestibility by beef cattle.
Linear: 0.03 Journal of Animal Science 79, 2268–2275. doi:10.2527/2001.
5.2 Quadratic: 0.60 7992268x
AOAC (2000) ‘Official methods of analysis.’ 17th edn. (Association of
5.1
Silage pH

Official Analytical Chemists: Arlington, VA)

5.0
4.9
4.8
4.7
0.0
24 48 72 96 120
Time (hours after aerobic exposure)
Fig. 2. pH after aerobic exposure of Mombasa grass silage treated
with increasing doses of lemongrass essential oil. Lemongrass
essential oil doses: : 0.00 (– ▪ –), 0.66 (– □ –), 1.33 (– ♦ –) and 2.00
(– ⋄ –) mL/kg.
present study. Lower accuracy decreases the power of the test.
Reduced fibre content and increased DM disappearance were
observed by Cantoia et al. (2020) using 2.00 mL of LEO per kg
of natural matter.
The temperature of silage after aerobic exposure showed
an unexpected pattern. However, LEO linearly reduced silage
pH after aerobic exposure. According to Kung et al. (2018),
yeast growth under aerobic conditions consumes lactic acid
and increases silage pH and temperature. Cantoia et al.
(2020) observed linearly reduced silage pH after aerobic
exposure when LEO was added up to 2 mL/kg as fed. This
effect was related to improved aerobic stability of sugarcane
silage due to the LEO effect on sporulation. The linear
reduction of silage pH after aerobic exposure reinforces that
the antifungal effect of LEO remained up to the aerobic
exposure.
LEO can be used as a grass silage additive to reduce
fermentation gas losses, NH3-N production, fibre content
and pH after aerobic exposure. However, it is essential to
pay attention to the moisture content of silage to avoid
excessive effluent losses.
Conclusion
Addition of LEO to Mombasa grass ensiling up to 2.00 mL per
kg (almost 7.5 mL/kg DM) reduces NH3-N concentration, gas
losses, ADF concentration and pH after aerobic exposure.
However, it is not sufficient to improve DM recovery,
in vitro disappearance or the nutritive value of grass silage.
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Campus Regional de Umuarama, Maringá State University, Umuarama 87502-970, Brazil.
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