Lemongrass essential oil reduces whole-plant sorghum silage gas losses and does not affect silage in vitro degradation

Abstract

Lemongrass essential oil (LEO) has been evaluated as a silage additive to improve silage fermentation and reduce fermentative losses. The present study aimed to evaluate the effects of increasing levels of LEO on whole-plant sorghum silage (WPSS) fermentation profile, fermentation losses, chemical composition, dry matter (DM) and neutral detergent fibre (NDF) in vitro degradation and aerobic stability. Five cultivars and fifty experimental silos were used to evaluate the following LEO levels: 0, 1, 2, 3 and 4 ml kg⁻¹ DM of WPSS. The material was compacted (650 kg m⁻²) and the silos were sealed, weighed and stored at room temperature for 167 days. The addition of LEO linearly decreased butyric acid content and the ratio between lactic and acetic acids. Intermediate levels of LEO increased NH₃-N and reduced lactic and acetic acids content. LEO linearly decreased silage gas losses. However, LEO did not affect total silage losses and DM recovery. Intermediate levels of LEO addition decreased organic matter and crude protein in the WPSS. The addition of LEO did not affect other chemical composition parameters, DM and NDF in vitro degradation, and pH and temperature of the silage after aerobic exposure. Thus, LEO supply in WPSS reduces gas losses, butyric acid concentration, and the ratio between lactic and acetic acids. However, LEO does not improve the chemical composition, in vitro degradation, and aerobic stability of WPSS.

Introduction

Maize silage is the main forage source in the Brazilian dairy cows' systems (Bernardes and Rego, 2014) because it is palatable, important as an energy and highly digestible forage source (Scheler and Cavichioli, 2021). However, weather factors such as temperature, precipitation and solar radiation can affect the quality and productivity of whole-plant maize silage (Maldaner et al., 2014). Whole-plant grain sorghum silage (WPSS) can be an alternative forage source that has higher adaptability to warm and dry environments (Zegada-Lizarazu et al., 2012) and shows high productivity and energy content.

Although sorghum is a more productive crop than maize, losses can be high during silage fermentation (Pinho et al., 2015). Several studies have attempted to improve the silage fermentation process, but the conditions are not always adequate to guarantee sufficient silage quality (McDonald et al., 1991). Microbial inoculants are the main group of additives used to improve dry matter (DM) recovery (Muck et al., 2018) and the aerobic stability of sorghum silage (Thomas et al., 2013). Kung et al. (2003) evaluated a blend of essential oils (EO) as an additive in maize silage. EO are natural secondary metabolites extracted from plants (Benchaar et al., 2008) and are known to have antimicrobial properties (Calsamiglia et al., 2007). Despite the antimicrobial effects of known EO, few studies using these substances as additives for silage have been carried out (Besharati et al., 2020).

The ban on antibiotics used as growth promoters is negatively impacting the livestock sector (Laxminarayan et al., 2016). Innovative alternatives are needed to produce animal feed and help combat rising antibiotic resistance (Czaplewski et al., 2016). In a recent study of our research group, Cantoia et al. (2020) observed positive effects using 2 ml of lemongrass essential oil (LEO) per kg of sugarcane silage (as-fed) on DM recovery, nutritional value and aerobic stability. This study also showed decreased yeast and mould count in LEO-treated silages, which are considered one of the main factors responsible for ethanol production and fermentation losses. Kholif et al. (2021) observed improvements in rumen fatty acids and milk yield and better nutrient utilization efficiency in lactating ewes fed lemongrass. However, to the best of our knowledge, there is no study evaluating LEO addition during the ensiling of a less fermentable material, such as whole-plant sorghum silage (WPSS). Although fermentability of fresh material affects additives' effects on silage fermentation profile (Oliveira et al., 2017), we hypothesized that increasing doses of LEO would reduce fermentation losses, silage butyric acid concentration, and increase the aerobic stability of WPSS. The present study aimed to evaluate the effects of increasing LEO levels on silage fermentation profile, fermentation losses, chemical composition, in vitro degradation of DM and fibre, and pH and temperature after aerobic exposure.

Materials and methods

The present trial was conducted at the Itaqui Campus of the Federal University of Pampa (29.2° South, 56.6° West, and 57 m above sea level). Experimental procedures were previously approved by the Animal Welfare Ethics Committee from the Federal University of Pampa (approval number 042/2019).

Treatments, experimental design and ensiling

Fifty experimental silos were made in PVC tubes with 28 cm diameter and 25 cm height. A completely randomized design was used to evaluate increasing doses of LEO addition during whole-plant sorghum ensiling: 0 (control); 1, 2, 3 and 4 ml per kg of sorghum DM. Lemongrass EO was obtained from Quinarí (Ponta Grossa, Brazil) and the doses were defined to be lower than those evaluated by Cantoia et al. (2020).

Five plots (each 180 m²) were conventionally prepared, and sown using five different sorghum cultivars (Nusil 426^®, Taguá^®, Nucover 100^® and Qualysilos^® from Sementes Nuseed, Curitiba, Brazil and AG2501^®, from Agroceres, Rio Claro, Brazil) on 01 November 2019. Within each cultivar, two silos were prepared for each of five levels of LEO. The harvest was conducted on two subsequent days from each plot. Harvest commenced when the first cultivar (plot) reached 400 g kg⁻¹ of DM content, and in subsequent days, it was performed in other plots. Plants were harvested on 7 (Soft dough stage) to 8 (Hard dough stage) phenological stage (Rao et al., 2007). The harvests occurred from February 11–17, 2020 (105 ± 2.44 days after the seeding). Plants were harvested at 5 cm of height and processed in a stationary mill (GP 1500 ADI, Garthen, Navegantes, Brazil). Representative samples of each cultivar were collected for chemical analysis, in vitro assay, and particle evaluation according to Maulfair et al. (2011) (Table 1). Dried sand (5 kg) was positioned inside the silo in a layer below the silage to quantify effluents. The sorghum material for each silo was individually weighed, and LEO (or placebo) was added using a pipette. Then, the sorghum was manually mixed and compacted to 650 kg m⁻³ of bunker density, sealed, and stored with shelter from light and heat. The temperature was not controlled and averaged 19°C during the storage period.

Table 1. Chemical composition and particle size of whole-plant Sorghum bicolor (L) before the ensiling (n = 5)

^a Stardard deviation.

Data record and sampling

The silos were opened 168 ± 2.44 days after the ensiling. The extended period of storage occurred due to the COVID-19 pandemic condition. Silos were weighed before opening to assess the gas losses through the storage period. Once the silos were opened, the silage was completely removed from the silos; 5-cm of the top and bottom layer was discarded, and the silage was manually mixed to obtain samples for fermentation profile evaluation (500 g), chemical analysis and in vitro assay (500 g) and aerobic stability evaluation (3 kg).

After sampling, silage fluid was extracted using a hydraulic press, without any water addition. Silage pH was immediately evaluated using a bench pH metre (LUCA-210^®, Lucadema, São José do Rio Preto, Brazil). Fluid was frozen (−20°C) without acid addition, prior to subsequent analysis. Samples for chemical and in vitro analysis were dried in a forced-air oven at 60°C for 72 h, and processed in a knives mill (SL-31^®, Solab Científica, Piracicaba, Brazil) to pass through a 2 (in vitro assay) or 1-mm (chemical analysis) sieve before the storage until analysis. Samples for aerobic stability assay were packed in PVC pipes without compression and stored for 168 h in a temperature-controlled room (21.2 ± 2.27°C) (Wilkinson and Davies, 2012). The temperature at the centre of silage mass was evaluated every 12 h using a spit thermometer (K29-5030^®, Kasvi – Produtos Laboratoriais, Pinhais, Brazil). Silage pH was recorded every 12 h after 15-min of water homogenization (dilution rate 15 g: 100 ml; Kung et al., 1984).

Chemical analysis and in vitro assay

Silage fluid was thawed at room temperature, and centrifuged (500 × g for 15 min.) to remove solid contaminants. The supernatant was used to analyse ammonia (NH₃-N), ethanol, and organic acids. Ammonia-N was analysed using the Kjeldahl method (984.13, AOAC, 2000) without sample digestion. The concentration of lactic acid was assessed after sulphuric acid solubilization and heating (75°C for 2.5 min). Samples were cooled and heated at 90°C for 1.5 min., after the addition of a colour reagent (4-phenylphenol, Sigma Aldrich, St. Louis, USA) addition. Readings were performed in a spectrophotometer at 560 nm (Pryce, 1969). Ethanol and other organic acids were evaluated using a gas chromatographic method. The sample was acidified with ortho-phosphoric acid (1.8 ml sample: 0.2 acid) and injected in a gas chromatograph (GC-2010 plus chromatograph, Shimadzu, Barueri, Brazil), equipped with an auto-sampler AOC-20i, capillary column Stabilwax-DA^™ (30 m, 0,25 mm ID, 0.25 μm df, Restek^©), and a flame ionization detector. It was used 1 μl of sample and 40:1 split ratio. Helium was the carrier gas and injection velocity was 42 cm s⁻¹. The injector and detector temperatures were 250 and 300°C, respectively, whereas the initial temperature of the column was 40°C. The temperature increased from 40 to 120°C at 40°C min⁻¹ rate, followed by increases from 120 to 180 and from 180 to 240°C at 10 and 120°C min⁻¹, respectively. Then, the temperature remained for 3 min at 240°C. Fatty acids were quantified based on the peaks areas, and qualifications were realized using GC solution v. 2.42.00 software.

Unfermented sorghum and silage samples (processed at 1-mm sieve) were analysed for DM (method 930.15; AOAC, 2000), ash (method 942.05; AOAC, 2000), crude protein (N × 6.25; Kjeldahl method 984.13; AOAC, 2000), ether extract (method 920.39; AOAC, 2000), acid detergent fibre and lignin (method 973.18; AOAC, 2000), and neutral detergent fibre (NDF) using thermal-stable alpha-amylase without sodium sulphite (Van Soest et al., 1991). Fibre contents were expressed including residual ash.

In vitro assay was performed according to Tilley and Terry (1963) and Holden (1999) methods. Samples (processed at 1-mm sieve) were placed in nonwoven fabric (5 × 5 cm and 100 g m⁻²; Casali et al., 2008) and incubated for 48-h in ruminal inoculum using an in vitro incubator (NL162^®, New Lab, Piracicaba, Brazil). The inoculum was prepared using ruminal fluid from two Dairy heifers maintained fed with Mombaça Guinea grass (Megathyrsus maximus) with no supplementation. Buffer was as described by McDougall (1948). After incubation, bags were washed in running water, and analysed for NDF content, as previously described.

Calculations and statistical analysis

Gas (GL, Eqn 1) and effluent losses (EL, Eqn 2) were calculated using the following equations (Jobim et al., 2007):

(1)$$GL\left( {\displaystyle{g \over {kg\; DM}}} \right) = \displaystyle{{WSWen\left( g \right)-WSWop\left( g \right)} \over {EDM\; \left( {kg} \right)}}$$

(2)$$EL\left({\displaystyle{g \over {kg\;DM}}} \right) = \displaystyle{{ESW_{op}( g ) -ESW_{en}( g ) } \over {EDM\;( {kg} ) }}$$

where WSW and ESW are whole and empty silos weight, respectively; en is weight at ensiling, whereas op is weight at silos opening, and EDM is ensiled DM. DM recovery is the ratio between DM obtained after storage and EDM.

Data were analysed using the PROC MIXED of SAS (version 9.4) according to the following model:

(3)$$Y_{ijk}\, = \,{\rm \mu }\, + \,LEO_i\, + \,C_j\, + \,LEO\, \times \,C_{ij}\, + \,e_{ijk}$$

With $e_{ij}\approx N\; \left( {0,\; \sigma _e^2 } \right)$, where: Y_ijk is the observed value of the dependent variable; LEO_i is the fixed effect of LEO level (i = 1 to 5); C_j is the fixed effect of cultivar (j = 1 to 5); LEO × C_ij is the LEO and cultivar fixed interaction effect; e_ijk is the random residual error; N stands for Gaussian distribution; $\sigma _e^2$ is the error variance. The LEO level effect was studied using polynomial regression: it evaluated the linear, quadratic, and non-quadratic (cubic) effect of LEO on evaluated variables.

Temperature and pH data obtained after aerobic exposure were analysed using the following model:

(4)$$Y_{ijkl} = \mu + LEO_i + C_j + \; LEO \times C_{ij} + \; \omega _{ijk} + \; T_l + \; LEO \times T_{il}\; + \; C \times T_{\,jl}\; + \; LEO \times C \times T_{ijl} + \; e_{ijkl}$$

with ω_ij ≈ N (0, $\sigma _\omega ^2$); and e_ijkl ≈ MRN (0, R); where $Y_{ijkl}$ is the observed value of the dependent variable; μ, $LEO_i$, C_j, LEO × C_ij, and N were previously defined; $\omega _{ijk}$ is the error associated with parcels (silos); $T_l$ is the fixed effect of time after aerobic exposure; $LEO \times T_{il}$, $C \times T_{jl}$, and $LEO \times C \times T_{ijl}$ are the fixed interaction effects between previously defined effects; $e_{ijkl}$ is the experimental error; $\sigma _\omega ^2$ is the variance associated with parcels (silo); MRN: stands for multivariate analysis with approximately Gaussian distribution; R is the matrix of variance and covariance due to repeated measures. The following matrices were evaluated according to the Bayesian method: CS, CSH, AR, ARH, TOEP, TOEPH, UN, FA, ANTE. Treatment effect was decomposed when P ≤ 0.10. Significance was declared at P ≤ 0.05.

Results

Fermentation profile

Utilization of LEO linearly decreased (P ≤ 0.01) butyric acid concentration and lactic to acetic acids ratio and linearly increased (P = 0.05) propionic acid concentration in WPSS (Table 2). Except for propionic acid silage concentration, there was no LEO and cultivar interaction effect (P ≥ 0.07) on the silage fermentation profile. In addition, LEO quadratically affected (P ≤ 0.05) silage NH₃-N, lactic, and acetic acids concentrations. Intermediary levels of LEO increased NH₃-N and reduced acetic and lactic acids concentrations in relation to control and upper level (4 ml kg⁻¹ DM). However, treatments showed no effects (P ≥ 0.12) on silage pH and concentrations of ethanol and branched-chain fatty acids.

Table 2. Silage fermentation profile and losses of WPSS containing increasing levels of LEOs

^a Increasing levels of LEO in WPSS: 0, 1, 2, 3 and 4 ml kg⁻¹ DM.

^b Probabilities: LEO effect (Treat.), linear (Lin.), quadratic (Qua.); and non-quadratic/cubic (Cub.) of LEO.

^c Branched-chain fatty acids.

Fermentative losses and DM recovery

There was no LEO and cultivar interaction effect (P ≥ 0.17) on fermentation losses and DM recovery. The addition of LEO linearly decreased (P ≤ 0.01) gas losses of silage. Lemongrass EO did not affect (P ≥ 0.89) effluent and total losses. Therefore, treatments showed no effect (P = 0.44) on DM recovery.

Chemical composition and in vitro degradation of DM and NDF

There was no LEO and cultivar interaction effect (P ≥ 0.06) on silage chemical composition and in vitro degradation (Table 3). The intermediate levels of LEO addition increased (P ≤ 0.03) the organic matter and crude protein of the silage. However, increasing levels of LEO did not affect (P ≥ 0.41) NDF, acid detergent fibre, acid detergent lignin, non-fibrous carbohydrates and ether extract content of the silage. Therefore, LEO had no effect (P ≥ 0.11) on in vitro degradation of DM and NDF.

Table 3. Chemical composition and in vitro degradation of WPSS containing increasing levels of LEOs

^a Increasing levels of LEO in WPSS: 0, 1, 2, 3 and 4 ml kg⁻¹ DM.

^b Probabilities: LEO effect (Treat.), linear (Lin.), quadratic (Qua.); and non-quadratic/ cubic (Cub.) of LEO.

Aerobic stability

After aerobic exposure, the addition of increasing levels of LEO did not affect (P ≥ 0.32) the silage pH and temperature regardless of the cultivar (P ≥ 0.13) and evaluation period (P ≥ 0.20) (Figs 1 and 2).

Fig. 1. pH after aerobic exposure of WPSS containing increasing levels of LEOs.

Fig. 2. Temperature after aerobic exposure of WPSS containing increasing levels of LEOs.

Discussion

We hypothesized that LEO addition during WPSS ensiling could reduce fermentative losses and butyric acid silage content and increase silage in vitro degradation and aerobic stability of silage, based on previous studies in sugarcane silage (Cantoia et al., 2020). Although LEO reduced silage gas losses and butyric acid concentration, it did not affect DM recovery, in vitro degradation, and silage temperature and pH after aerobic exposure.

In the present study, we found a quadratic effect of LEO on NH₃-N concentration, once silages containing 1 ml kg⁻¹ of LEO had higher NH₃-N concentration than control-silage. In a previous study of our research group (Cantoia et al., 2020), LEO increased NH₃-N in sugarcane silage. Microbial and plant enzymes are the main accountable for protein solubilization and proteolysis (Junges et al., 2017). In addition, NH₃-N and butyric acids are produced in high humidity silages by clostridial fermentation (Kung et al., 2018). Studies evaluating LEO in beef cattle diet observed a reduction in NH₃-N in vivo (Wanapat et al., 2008) and in vitro (Nanon et al., 2014) and associated this with a negative effect of LEO on rumen ammonia bacterial production. In another study by our research group, Garcia et al. (2022) observed a reduced NH₃-N concentration in Guinea grass silage when LEO was added during ensiling. Ammonia-N has been associated with increased silage pH observed in LEO-treated silages: reduced pH could inhibit clostridial growth, showing a negative effect on NH₃-N concentration (Kung et al., 2018). In the current study, LEO had no effect on silage pH and, therefore, it was possible to confirm that LEO inhibits WPSS proteolysis, once LEO reduces NH₃-N and butyric acid content on silage.

As observed in NH₃-N concentration, LEO showed a quadratic effect on silage CP and OM content. Increased protein solubilization increases N losses through the effluents, resulting in a lower CP content in the silage. As observed by Chaves et al. (2008), EO' effects on silage fermentation and aerobic stability are greatly affected by supplying level. On the other hand, LEO effect on OM content has limited implications on silage nutritional value.

Besides reported effects on NH₃-N and butyric acid concentrations, intermediary levels of LEO reduced the acetic and lactic concentrations on WPSS. Evaluating increasing levels of cumin EO in alfalfa silage, Turan and Önenç (2018) also observed a quadratic effect on NH₃-N and organic acids production. According to those authors, the inhibition of lactic acid bacteria metabolism reduces organic acids production and proteolysis. However, increasing LEO levels linearly decreased the lactic to acetic acid ratio. This effect agrees with heterolactic fermentation, as observed using second-generation (heterolactic) microbial inoculants (Arriola et al., 2011). Besides linear negative of LEO on both acids production, Cantoia et al. (2020) also observed a lactic to acetic ratio of 1.62 in control and 1.36 using 3 ml of LEO per kg of sugarcane. Heterolactic fermentation normally has a more positive effect on high fermentable substrates, as sugarcane evaluated by Cantoia et al. (2020) than WPSS evaluated in the present study (Oliveira et al., 2017).

Although heterolactic fermentation results in higher water and CO₂ production than homolactic fermentation (Muck, 2010), the addition of LEO linearly reduced silage gas losses. Furthermore, possible increased gas production due to heterolactic fermentation largely reduced butyric acid content. According to Kung et al. (2018), silages with higher clostridial growth have high concentrations of fibre and low DM digestibility because much of the readily available soluble nutrients have been degraded (Mills and Kung, 2002). This degradation seems to increase gas losses of low-LEO treated silages. Besides the reduced lactic to acetic acid ratio, LEO increased propionic acid concentration in the present study. Inoculation with propionic acid bacteria has been used to increase propionic acid and increase aerobic stability by inhibiting yeast and mould growth (Filya et al., 2004). Although LEO did not affect WPSS aerobic stability, increased propionic concentration contributed to decreased fermentation gas losses in the present study.

Besharati et al. (2021) evaluated increasing levels of lemongrass seed essential oil on alfalfa silage fermentation profile and in vitro de gradation kinetics. Intermediary levels evaluated in that study (60 ml /kg⁻¹ DM) increased the potential DM degradation. However, in the present study there were no treatment effects on DM recovery, silage fibre concentration, and in vitro degradation of DM and NDF. It is possible to infer that LEO positive effects on silage fermentation and gas losses were slight to affect these variables. Other questions, such as effluent losses and fluidification of DM, could affect DM recovery and result in a lack of LEO impact on this variable. Although increased acetic acid content of silage can reduce feed intake (Steen et al., 1998), and butyric acid has been associated with decreased animals' performance (Scherer et al., 2015), the absence of LEO effect on DM recovery, fibre content, and in vitro degradation limits its application in practical conditions, once these variables are the most associated with the financial viability of the additive.

The addition of LEO showed no effect on WPSS temperature and pH after aerobic exposure. A linearly reduced lactic to acetic ratio could lead us to expect increased aerobic stability of WPSS: acetic acid inhibits yeast and mould growth after aerobic exposure, whereas lactic acids serve as the substrate for these microorganisms' growth (Danner et al., 2003). However, butyric has been associated with extended aerobic stability (Kung et al., 2018). Consequently, LEO did not improve the aerobic stability of WPSS in the present study. Kung et al. (2003) evaluated a blend of essential oil addition during whole maize plant ensiling. Besides positive effects observed on in vitro ruminal fermentation and animal performance, EO had no effects on silage fermentation and aerobic stability. Similarly, LEO altered WPSS silage fermentation and has limited effects on silage nutritional value and aerobic stability.

As previously mentioned, fermentability is one of the main factors influencing the effects of EO on silage parameters (Chaves et al., 2008). EO could be considered inhibitors of silage fermentation by their negative effects on microbial growth. Considering the lower fermentability of WPSS compared to sugarcane silage, we prefer to evaluate lower LEO doses than the optimal doses recommended by Cantoia et al. (2020). Low-fermentability material response is better when treated with fermentation promoters rather than inhibitors. It also explains the lower effects of LEO on Guinea Grass (Garcia et al., 2022) and WPSS (current study) when compared to sugarcane silage (Cantoia et al., 2020).

Conclusion

LEO reduces WPSS gas losses, butyric acid concentration, and the ratio between lactic and acetic acids. However, LEO does not improve silage chemical composition, in vitro degradation, or aerobic stability.