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Article

Evaluation of Plant Essential Oils as Natural Alternatives to Monensin in In Vitro Ruminal Fermentation

by
Amelia Barbosa Lima
1,
Kemmily Lima de Almeida
1,
Bruna Eduarda Teixeira de Lima
1,
Khalid Haddi
2,
Ludmila Couto Gomes Passetti
1,
Gustavo Leão Rosado
1 and
Cláudia Braga Pereira Bento
1,*
1
Institute of Agricultural Sciences, Universidade Federal dos Vales do Jequitinhonha e Mucuri, Unaí 38610-000, Brazil
2
Department of Entomology, Universidade Federal de Lavras, Lavras 37200-900, Brazil
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(12), 693; https://doi.org/10.3390/fermentation11120693 (registering DOI)
Submission received: 28 October 2025 / Revised: 10 December 2025 / Accepted: 12 December 2025 / Published: 14 December 2025
(This article belongs to the Special Issue Ruminal Fermentation: 2nd Edition)
Versions Notes

Abstract

Extensive growth promoter use in livestock production has raised concerns about their role in selective pressure on resistant microorganisms, driving interest in natural alternatives such as essential oils (EOs). This study aimed to evaluate the effects of tea tree, holy wood, and citronella EOs on in vitro ruminal fermentation. The study follows a completely randomized design with the following five treatments: control, monensin (5 μM), tea tree EO (50 mg/L), holy wood EO (50 mg/L), and citronella EO (50 mg/L), each conducted in triplicate. Incubations were performed at 39 °C for 48 h in the rumen fluid collected from fistulated cattle fed a 20:80 forage-to-concentrate diet. Notably, EOs exhibited no significant effects on pH, microbial protein production, total volatile fatty acids, or in vitro dry matter digestibility (p > 0.05). Tea tree and holy wood EOs enhanced deamination activity, and all treatments increased ammonia concentration compared with that in the control. Monensin treatment increased acetate concentration and reduced in vitro neutral detergent fiber digestibility; holy wood EO exhibited a similar trend. Altogether, the findings of this study suggest that EOs can selectively modulate the ruminal microbiota, influencing nitrogen metabolism and fermentation patterns without impairing rumen stability.

1. Introduction

Ruminants play an essential role in global food security by providing food, generating employment, and contributing to the world economy [1]. Reportedly, the global population is expected to reach 9.15 billion by 2050, intensifying the demand for animal protein [2,3]. However, livestock farming remains debated owing to its environmental impact, particularly on climate change [4]. Ruminants contribute to approximately 14.5% of anthropogenic greenhouse gas (GHG) emissions, particularly methane, carbon dioxide, and nitrous oxide (N2O), which exacerbates global warming [5,6].
Beyond environmental concerns, these emissions reflect energy and protein losses, highlighting compromised production efficiency [7]. For example, N2O emissions reflect dietary protein loss, indicating suboptimal animal production and nitrogen utilization, whereas methane emissions represent a marked loss of gross energy from the ruminant diet [8,9]. Therefore, reducing GHG emissions and meeting climate goals are essential [10].
To date, many nutritional strategies have been explored to address the challenge of GHG emissions, particularly the use of ruminal fermentation-modulating additives [11,12,13]. Among them, ionophores have proven effective in reducing GHG emissions, improving digestibility, and preventing digestive disorders, leading to an overall positive impact on productivity and economics [14]. However, their antimicrobial characteristics have raised concerns regarding the development of resistant bacteria. Consequently, their use as food additives has been banned in some European Union countries since 2006, restricted in countries such as Argentina, and remains under review in many others [15,16].
This has led to growing interest in plant-derived natural additives as promising alternatives, especially essential oils (EOs) [17]. EOs are volatile compounds derived from plant secondary metabolism and exhibit antimicrobial activities through various mechanisms, including cell wall degradation, membrane permeability alteration, cytoplasmic coagulation, and enzyme inactivation [18]. In the rumen, their action has been reported to be more pronounced on Gram-positive bacteria, which leads to further changes in ruminal microbial activity and fermentation dynamics [19,20].
Reportedly, EOs can reduce protein degradation and subsequent ammonia (NH3) release in the rumen [21,22]. They also influence the volatile fatty acid (VFA) profile, reduce the acetate–propionate (A:P) ratio, and increase propionate production, which represents a more efficient energy generation approach [23]. Additionally, EOs can suppress methanogenesis-related microbial metabolic pathways, thereby contributing to the mitigation of methane emissions [21]. Moreover, evidence suggests that they can improve fiber degradation and nutrient digestibility [24].
Based on this, including EOs in the diet of ruminants may optimize ruminal fermentation, increase energy- and nutrient-utilization efficacy, improve microbial protein production, and help alleviate digestive disorders. Beyond their zootechnical and environmental impact, the use of EOs as natural antibiotic substitutes may also increase consumer acceptance and strengthen the positive image of animal products [25].
Despite these advantages, studies reporting the effects of EOs present notable inconsistencies, which are often related to both the chemical diversity and the doses and combinations of EOs [26,27]. These inconsistencies necessitate further research to better understand the specific effects of EOs, derived from different sources, on ruminal fermentation. Hence, this study aimed to investigate EOs from tea tree (Melaleuca alternifolia), holy wood (Bursera graveolens), and citronella (Cymbopogon winterianus) to elucidate their effects on in vitro ruminal fermentation and nutrient digestibility. Additionally, monensin, a widely used polyether antibiotic in the feed of ruminant animals, was employed to compare the efficacy of EOs.

2. Materials and Methods

2.1. Procurement of EOs

The EOs of tea tree, holy wood, and citronella were commercially obtained in sealed 10 mL amber bottles from the WNF Indústria e Comércio Ltda (São Paulo, Brazil).

2.2. Phytochemical Characterization

Phytochemical identification of the components in the tea tree, holy wood, and citronella EOs was performed through gas chromatography (GC)–mass spectrometry using a QP 2010 Plus system (Shimadzu Corporation, Kyoto, Japan) equipped with a fused silica capillary column (30 m × 0.25 mm) and a DB-5 bonded phase (film thickness: 0.25 μm). Helium served as the carrier gas at a flow rate of 1.0 mL/min, and injector and detector temperatures were set at 220 °C and 240 °C, respectively. Samples (0.5 μL) were injected after being diluted in 1% hexane (Sigma-Aldrich®, St. Louis, MO, USA), using a split ratio of 1:100. The temperature program initiated at 60 °C, increased at a rate of 3 °C/min up to 240 °C, then by 10 °C/min until reaching the final temperature of 300 °C, where it was held for 7 min. The column pressure was maintained at approximately 71.0 kPa. The mass spectrometer operated at 70 eV ionization potential and a source temperature of 200 °C. The mass spectra were acquired in full scan mode (45–500 Da), with a scan speed of 1000 Da/s and an acquisition interval of 0.5 fragments/s. Data were obtained and processed using the Lab Solutions LC/GC Workstation 2.72 software (Shimadzu Corporation, Kyoto, Japan). Retention indices were calculated using a homologous series of n-alkanes (nC9–nC18) and the Van den Dool and Kratz equation, as previously described [28]. Compound identification involved comparing calculated retention indices with literature values and matching the obtained mass spectra against the FFNSC 1.2, NIST 107, and NIST 21 libraries. Quantitative analysis was carried out using GC with a flame ionization detector on a GC-2010 system (Shimadzu Corporation, Kyoto, Japan), under identical conditions to the qualitative analysis described above, except for the detector temperature (300 °C) and the use of acetylene as carrier gas. The relative percentages of each compound were determined using the area normalization method.

2.3. In Vitro Treatments and Incubations

Ruminal fluid was collected from three fistulated male cattle (6 years old, weight: 615 ± 20 kg) at the Santa Paula Experimental Farm, Universidade Federal dos Vales Jequitinhonha e Mucuri, Unaí campus. The cattle grazed freely on Marandu grass (Urochloa brizantha) and received freshwater and salt ad libitum. Sampling was performed 2 h after feeding, followed by the filtering of the ruminal fluid through four layers of gauze and transfer to the laboratory in thermal containers. The study employed a completely randomized design with the following five treatments: (1) control (no additive); (2) positive control (5 µM monensin); (3) tea tree EO; (4) holy wood EO; and (5) citronella EO, with each treatment performed in triplicate (total of 15 bottles per treatment). The experiment was repeated independently three times (a total of 45 bottles). EOs were tested at a concentration of 50 mg/L of ruminal fluid. For in vitro incubation, 350 mg of a concentrate diet–corn silage mixture was used, which consisted of 280 mg of the concentrate diet with 22% crude protein (CP) and 70 mg of corn silage, yielding a 20:80 forage-to-concentrate ratio based on dry matter (DM).
The chemical composition of the feed mixture was analyzed for DM [29] (method 934.01), organic matter [29] (method 930.05), ash [29] (method 942.05), and total nitrogen [29] (method 981.10). CP was by multiplying total nitrogen to the factor 6.25. Neutral detergent fiber (NDF) was determined following the approach of Mertens [30], without the addition of sodium sulfite and thermostable α-amylase (Table 1).
The in vitro rumen incubations were conducted under anaerobic conditions in 50 mL penicillin flasks with 35 mL of ruminal fluid (pH = 6.7 ± 0.15), 350 mg of diet, and 10 µL of the assigned treatment, along with agitation at 160 rpm and 39 °C for 48 h.

2.4. Determination of the Specific Activity of Deamination, NH3 Concentration, and Microbial Protein Production

Herein, 1 mL samples were collected at 0, 6, and 24 h of incubation to determine NH3 concentration using the colorimetric method of Chaney and Marbach [31]. Absorbance was measured at 630 nm using a spectrophotometer (Thermo Scientific, Waltham, MA, USA), and ammonium chloride was used as the standard. The NH3 concentration (in mM) was calculated as the difference between the total NH3 at 24 h post-incubation and that present at 0 h.
Additionally, 0.5 mL samples were collected at 0, 6, and 24 h of incubation to determine microbial protein (average content produced at 0, 6, and 24 h) using the Bradford method [32], with lysozyme serving as the standard.
The specific activity of deamination (SAD) was calculated as the difference in NH3 concentration (mM) at 0 and 6 h, divided by the average microbial protein concentration (mg/L, averaged between 0 and 6 h) and the incubation time (min).

2.5. Determination of Organic Acids

After 24 h of incubation, samples were collected for determining VFAs and their molar proportions employing high-performance liquid chromatography. Ruminal fluid samples (1.0 mL) were centrifuged (12,000× g, 10 min), and the cell-free supernatant was processed as described by Siegfried et al. [33]. VFA analysis was performed using a Dionex Ultimate 3000 Dual chromatograph (Dionex Corporation, Sunnyvale, CA, USA) coupled to a Shodex RI-101 refractive index detector and a Phenomenex Rezex ROA ion-exclusion column (300 × 7.8 mm) (Phenomenex Inc., Torrance, CA, USA) maintained at 45 °C. The mobile phase consisted of 5.0 mM sulfuric acid at a flow rate of 0.7 mL/min. The following organic acids were used for calibrating the standard curve: acetic, propionic, valeric, isovaleric, isobutyric, and butyric acids, with all acids being prepared with a final concentration of 10 mmol/L, except isovaleric acid (5 mmol/L) and acetic acid (20 mmol/L).

2.6. Determination of In Vitro Digestibility of Dry Matter and Neutral Detergent Fiber

After 48 h incubation, the material was filtered in crucibles and washed thrice with hot water. The crucibles were oven-dried at 105 °C to a constant dry weight and used to calculate in vitro dry matter digestibility (IVDMD). Afterward, the filtering crucibles were placed in universal flasks with 70 mL of neutral detergent solution and autoclaved for 1 h at 105 °C. After autoclaving, crucibles were rinsed with hot water to remove the neutral detergent solution, followed by a final rinse with 15 mL of pure acetone. Next, they were oven-dried at 105 °C to a constant weight. The final weight was used to determine in vitro neutral detergent fiber digestibility (IVNDFD).

2.7. Statistical Analyses

The experiment was structured as a completely randomized design, with three replicates per treatment, and repeated independently three times. The mathematical model used was as follows:
Yij = μ + Ai + eij
where μ denotes the mean of the treatments; Ai represents the effect of the A treatment, ranging from 1 to 5; and eij denotes random error. Data for pH, SAD, NH3 concentration, microbial protein, total VFA and fractions, IVDMD, and IVNDFD were subjected to analysis of variance to compare treatments, with means compared using Tukey’s test at a 5% significance level using the SISVAR software (Version 5.8) [34].

3. Results

Phytochemical analysis of the three evaluated EOs revealed terpene-rich profiles (Table 2). Citronella EO was rich in oxygenated monoterpenes, primarily geraniol (28.79%), citronellal (24.33%), and citronellol (16.49%), accounting for approximately 70% of its composition. Tea tree EO was characterized primarily by monoterpenes, with terpinen-4-ol (54.39%), γ-terpinene (16.93%), p-cymene (9.62%), and α-terpinene (5.93%) as the major constituents. Holy wood EO contained four main components, with limonene (61.54%), α-phellandrene (35.00%), and menthofuran (1.31%) belonging to the monoterpene class and the sesquiterpene germacrene D (1.08%).
Notably, supplementation with EO or monensin did not affect pH values (p = 0.957) after 48 h of in vitro incubation, and it remained stable between 5.41 and 5.43 (Table 3). Similarly, microbial protein showed no significant differences among treatments (p > 0.05). Although no statistical difference was observed, citronella EO increased microbial protein (1322.07 mg/mL) compared with that in the other groups (Table 3).
Tea tree and holy wood EOs increased (p < 0.01) the SAD when compared to both the control and monensin treatments (Table 3). All EO and monensin treatments markedly increase the NH3 concentration (p = 0.001) compared with that in the control (Table 3).
No significant differences were observed for total volatile fatty acid (VFA) concentration following supplementation with monensin and different EOs (tea tree, citronella, and holy wood) (p = 0.882) (Table 4). Monensin supplementation resulted in a higher molar proportion of acetic acid (73.45 mol/100 mol; p = 0.008) and the lowest proportions of butyric acid (6.02 mol/100 mol; p < 0.01) and valeric acid (0.52 mol/100 mol; p = 0.029) compared with those in the control and EO groups (Table 4). There were no significant differences between treatments for isobutyric (p = 0.452), isovaleric (p = 0.285), and propionic (p = 0.056) acids, along with the A:P ratio (p = 0.090).
No intergroup differences (p = 0.183) were observed for IVDMD, with values ranging from 65.07% (control) to 62.65% (monensin). However, IVNDFD differed among treatments (p < 0.01) as follows: IVNDFD in both monensin (41.18%) and holy wood EO (36.83%) treatment significantly decreased (p < 0.01) compared with that in the control (50.42%) and tea tree EO (49.00%) treatments. In contrast, citronella EO (43.99%) showed an intermediate value that did not significantly differ (p > 0.05) from other treatments (Table 5).

4. Discussion

EOs constitute a complex group of plant secondary metabolites with a highly variable composition [35], which makes it challenging to define their mode of action against microorganisms, ultimately limiting the understanding of these oils in the ruminal environment [21]. In the present study, three EOs, with distinct phytochemical compositions, were evaluated as a potential alternative for monensin, aiming to determine their effects on ruminal fermentation in vitro.
Ruminal pH is an important parameter that indicates the internal homeostasis of the ruminal environment [36]. Herein, supplementation with EOs and monensin did not affect ruminal fluid pH after 48 h of in vitro incubation compared with that in the control. This pH stability suggests that ruminal functions were maintained under stable conditions, suggesting that the components in the evaluated EOs did not significantly impact acid production or ruminal pH [37].
Considering the diet composition with a high concentrate content (20:80 forage-to-concentrate ratio), which typically lowers ruminal pH owing to increased VFA production, the EO treatments successfully maintained pH levels similar to those observed with monensin. Monensin is widely used in cattle feedlots primarily to prevent pH from reaching critically acidic levels [38].
Similar findings where EO supplementation preserved ruminal pH comparable to monensin have been reported, reinforcing the effect of EOs on this parameter [39,40,41]. For instance, Tawab et al. [42] reported no significant changes in ruminal pH in vitro when using orange EO rich in monoterpenes such as limonene and α-pinene, which were also present in the EOs evaluated in the present study. Additionally, the lower total VFA concentration in EO treatments may have contributed to ruminal pH stability.
Monensin and EO supplementation did not alter total VFA concentration in ruminal fluid, indicating that the fermentative activity was not compromised. This aligns with the present literature suggesting that EO compounds, despite exhibiting selective antimicrobial effects, can preserve total VFA production at moderate concentrations [4,43,44].
Monensin increased the molar proportion of acetic acid while reducing butyric and valeric acid proportions. Although monensin typically decreases acetate and increases propionate production [4,21,45,46], the pattern observed in this study may be attributed to a possible synergistic interaction between the ionophore and the acidic pH observed in vitro (approximately 5.4), indicative of subacute ruminal acidosis. Studies show that under low pH, propionate-producing Gram-negative bacteria exhibit greater resistance to monensin, whereas Gram-positive acetate-producing populations may be less inhibited [47]. Additionally, recent evidence suggests that acidic pH favors acetate formation through modulating fermentation thermodynamics [48]. Therefore, the observed fermentation profile possibly results from the combined effects of monensin and acidic conditions of the medium rather than the sole impact of microbial adaptation by Gram-positive bacteria.
In contrast, EO treatments did not significantly alter individual VFA profiles, suggesting that the monoterpenes present in EOs (including terpinen-4-ol, citronellal, and limonene) did not exert selective antimicrobial effects in the fermentative microbiota at tested concentrations. This lack of selectivity may explain the unchanged A:P ratio and subsequent maintenance of a fermentation profile associated with effective fiber digestibility [44].
Additionally, microbial protein synthesis showed no significant differences between treatments, indicating that none of the tested additives stimulated the growth of microorganisms involved in microbial protein production. However, citronella EO did increase microbial protein concentration (1322.07 mg/mL) compared to the other treatments, albeit non-significantly, suggesting a possible modulatory effect on ruminal microbiota.
Evidence suggests that limonene, a constituent of citronella EO, can reduce populations of hyper-ammonia-producing bacteria, creating a more favorable environment for microbial protein synthesis [49]. Nonetheless, this information is controversial because geraniol, the major compound in citronella EO, has been associated with increased ruminal NH3 concentrations in some studies, which potentially diminishes nitrogen utilization efficiency and increases environmental pollution [49,50,51].
Quantifying NH3 concentration is a critical indicator of nitrogen utilization efficiency during in vitro bioassays, as 60–80% of nitrogen incorporated by ruminal microbes originates from NH3 [4]. In this study, the SAD value increased significantly with tea tree and holy wood EOs compared with that of the control and monensin. This increase may reflect selective enrichment of microorganisms that preferentially deaminate, leading to accumulation of free NH3 in the ruminal environment, which, in turn, increases SAD [52,53]. This selection may be associated with antibacterial effects of compounds such as terpinen-4-ol (tea tree EO) and limonene (holy wood EO) [54]. This is consistent with the finding that monoterpenes can stimulate ruminal proteolysis, promoting dietary protein degradation and elevated NH3 release into the ruminal environment [55].
The observed increase in NH3 concentration with EOs and monensin treatments compared with that in the control, without a corresponding increase in microbial protein production suggests inefficient use of ammoniacal nitrogen in microbial protein synthesis [56]. This inefficiency causes nutritional and energy losses, as excess NH3 requires metabolism for degrading dietary protein and absorption by the ruminal wall, highlighting inefficiency in the utilization of dietary nitrogen and potential environmental impacts [57].
Literature reports conflicting findings on NH3 production in the ruminal environment, as some studies indicate an increase in NH3 concentration with the use of EOs [49,58]. This increase in ruminal NH3 can elevate urinary nitrogen excretion, primarily via urea, which, once deposited in the soil, is rapidly converted to NH4+ by microorganisms and then into NO3− through nitrification; under low-oxygen conditions, it further undergoes denitrification, releasing N2O [59,60].
In contrast, some studies have not reported any significant effects [4,61], whereas others observed a reduction in NH3 concentration [62,63]. Overall, these results reinforce the hypothesis that, although EOs can modulate ruminal fermentation, their effects on nitrogen metabolism depend on the balance between protein degradation and nitrogen incorporation by microorganisms [44].
EOs may exhibit adverse effects on ruminal fermentation owing to broad and nonspecific antimicrobial activity [17]. For instance, Cobellis et al. [21] reported that while EOs do not affect fiber degradation, they can inhibit degradation of easily degradable substrates such as proteins and starch by limiting colonization and digestion of these substrates by amylolytic and proteolytic bacteria.
In this study, neither monensin nor EO addition induced any changes in IVDMD. This is consistent with the findings of previous studies, which report that, despite their ruminal fermentation-modulating activity, both monensin and phytochemicals, such as terpenes, do not always affect DM digestibility when used at moderate concentrations [39,44].
However, IVNDFD showed significant differences between the evaluated treatments in the present study. Holy wood EO (36.83%) and monensin (41.18%) exhibit marked IVNDFD reduction compared with that in the control (50.42%) and tea tree EO (49.00%). This reduction was attributed to the inhibitory effects of both EOs and monensin on cellulolytic microbial populations, such as Ruminococcus flavefaciens and Fibrobacter succinogenes, which are key species for fiber degradation [11,64]. Monensin, as an ionophore, decreases these bacterial populations, thereby limiting plant cell wall degradation [48]. The IVNDFD reduction observed with holy wood EO may stem from its potent antimicrobial compounds, such as limonene and α-terpineol, which have been shown to negatively affect the population of fibrolytic microorganisms [39]. Cobellis et al. [43] reported similar results regarding IVNDFD in their study, where they noted a significant reduction in NDF digestibility following oregano and rosemary EO supplementation. In contrast, tea tree EO maintained IVNDFD comparable to that in the control, indicating a less selective antimicrobial effect that preserves cellulolytic microbial activity [65].
Additionally, citronella EO exhibited an intermediate IVNDFD value (43.99%) without statistical difference from other treatments, suggesting a more balanced or less intense antimicrobial action that partially preserves the cellulolytic activity. Previous studies have shown that, depending on the dose used, citronellal, the second most abundant compound in citronella EO, exerts moderate effects on ruminal bacteria [44].

5. Conclusion

The findings of this study showed that tea tree, holy wood, and citronella EOs did not affect ruminal pH, microbial protein, total VFA, or IVDMD. However, they did increase SAD and NH3 concentration. Furthermore, holy wood EO (as well as monensin) resulted in a significant reduction in IVNDFD. In conclusion, these results demonstrate that the evaluated EOs act as selective and partial modulators of ruminal fermentation, notably influencing nitrogen metabolism and fiber degradation patterns without impairing the overall stability of the ruminal environment.

Author Contributions

A.B.L.: conceptualization, investigation, formal analysis, methodology, and writing—original draft; K.L.d.A.: formal analysis and methodology; B.E.T.d.L.: formal analysis and methodology; K.H.: conceptualization, resources and writing—review; L.C.G.P.: supervision, conceptualization, funding acquisition, and methodology; G.L.R.: conceptualization, methodology, and writing—review and editing; C.B.P.B.: conceptualization, supervision, resources, methodology, funding acquisition, project administration, and writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The animal study protocol was approved by the Animal Ethics Committee of the Universidade Federal dos Vales do Jequitinhonha e Mucuri (protocol no. 06/2022, approved on 17 August 2022).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

This research was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; Brasília, Brazil), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; Brasília, Brazil), the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG; Belo Horizonte, Brazil), and the Funding Authority for Studies and Projects (Finep).

Conflicts of Interest

The authors declare no conflicts of interest.

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Table 1. Chemical composition (percentage in dry matter) of the diet used in in vitro incubation.
Table 1. Chemical composition (percentage in dry matter) of the diet used in in vitro incubation.
ItemsCorn SilageConcentrate 1
DM 2 (% in DM)36.290.6
OM 3 (% in DM)96.093.2
CP 4 (% in DM)7.922.0
NDF 5 (% in DM)39.720.9
Ash (% in DM)3.96.7
1 Corn (37.8%), soybean meal (38.2%), sorghum (20%), and premix (4%) (calcitic limestone, sodium chloride, dicalcium phosphate, calcium iodate, magnesium oxide, zinc oxide, sodium selenite, cobalt sulfate, copper sulfate, manganese oxide, vitamin A, vitamin D3, vitamin E, and flower of sulfur) for lactating dairy cattle; 2 DM: dry matter; 3 OM: organic matter; 4 CP: crude protein; 5 NDF: neutral detergent fiber.
Table 2. Phytochemical composition (Relative Amount, %) of essential oils of citronella (Cymbopogon winterianus), tea tree (Melaleuca alternifolia), and holy wood (Bursera graveolens).
Table 2. Phytochemical composition (Relative Amount, %) of essential oils of citronella (Cymbopogon winterianus), tea tree (Melaleuca alternifolia), and holy wood (Bursera graveolens).
CompoundsTea Tree EO 1Citronella EO 1Holy Wood EO 1
Geranyl acetate-1.13-
γ-Cadinene-1.52-
β-Caryophyllene-2.43-
p-Cymene9.62--
1,8-Cineole1.62--
Citronellal-24.33-
Citronellol-16.49-
α-Phellandrene--35.00
Geranial-10.11-
Geraniol-28.79-
Germacrene D--1.08
Limonene1.652.6561.54
Menthofuran--1.31
Neral-8.16-
α-Pinene2.38--
α-Terpinene5.93--
γ-Terpinene16.93--
α-Terpineol4.28--
Terpinen-4-ol54.30--
Terpinolene2.12--
Minority compounds1.174.391.06
1 EO: essential oil.
Table 3. Effect of monensin and different essential oils on in vitro ruminal fermentation.
Table 3. Effect of monensin and different essential oils on in vitro ruminal fermentation.
ParametersControlMonensin
(5 µM)		Tea Tree EO 1Citronella EO 1Holy Wood EO 1p-Value
pH5.415.435.435.415.420.957
Microbial protein 21280.751198.561275.001322.07972.700.077
SAD 317.14 b22.30 b44.08 a21.34 b36.18 a<0.01
NH3 46.78 b14.45 a13.48 a13.12 a16.97 a0.001
1 EO: essential oil (50 mg/L). 2 Microbial protein (mg/L). 3 Specific activity of deamination (SAD; nmol of NH3/mg protein/min). 4 Ammonia concentration (NH3; mmol/L). Means followed by the same letter in the row do not differ at the 5% significance level according to Tukey’s test.
Table 4. Effect of monensin and different essential oils on the concentration of volatile fatty acids.
Table 4. Effect of monensin and different essential oils on the concentration of volatile fatty acids.
Organic Acids 1ControlMonensin
(5 µM)		Tea Tree EO 2Citronella EO 2Holy Wood EO 2p-Value
Total VFAs141.72138.91138.69137.65139.550.882
Acetic acid (A)67.13 b73.45 a66.96 b69.60 ab68.93 b0.008
Propionic acid (P)18.4518.7618.4516.6517.470.056
Butyric acid11.88 a6.02 c11.75 ab11.11 ab10.89 b<0.01
Isobutyric acid0.640.450.670.740.800.452
Valeric acid0.90 a0.52 b0.90 a0.83 ab0.91 a0.029
Isovaleric acid0.980.791.261.030.980.285
A:P3.643.913.634.173.940.090
1 Total VFAs (mmol/L), acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid (mol/100 mol). 2 EO: essential oil (50 mg/L). Means followed by the same letter in the line do not differ at a 5% significance level according to the Tukey test.
Table 5. Effect of monensin and different essential oils on in vitro dry matter digestibility and in vitro neutral detergent fiber digestibility.
Table 5. Effect of monensin and different essential oils on in vitro dry matter digestibility and in vitro neutral detergent fiber digestibility.
ParametersControlMonensin
(5 µM)		Tea Tree EO 1Citronella EO 1Holy Wood EO 1p-Value
IVDMD65.0762.6563.7764.5663.930.183
IVNDFD50.42 a41.18 b49.00 a43.99 ab36.83 b<0.01
1 EO: essential oil (50 mg/L). Means followed by the same letter in the line do not differ at a 5% significance level by the Tukey test. IVDMD = in vitro dry matter digestibility; IVNDFD = in vitro neutral detergent fiber digestibility.
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Lima, A.B.; de Almeida, K.L.; Lima, B.E.T.d.; Haddi, K.; Passetti, L.C.G.; Rosado, G.L.; Bento, C.B.P. Evaluation of Plant Essential Oils as Natural Alternatives to Monensin in In Vitro Ruminal Fermentation. Fermentation 2025, 11, 693. https://doi.org/10.3390/fermentation11120693

AMA Style

Lima AB, de Almeida KL, Lima BETd, Haddi K, Passetti LCG, Rosado GL, Bento CBP. Evaluation of Plant Essential Oils as Natural Alternatives to Monensin in In Vitro Ruminal Fermentation. Fermentation. 2025; 11(12):693. https://doi.org/10.3390/fermentation11120693

Chicago/Turabian Style

Lima, Amelia Barbosa, Kemmily Lima de Almeida, Bruna Eduarda Teixeira de Lima, Khalid Haddi, Ludmila Couto Gomes Passetti, Gustavo Leão Rosado, and Cláudia Braga Pereira Bento. 2025. "Evaluation of Plant Essential Oils as Natural Alternatives to Monensin in In Vitro Ruminal Fermentation" Fermentation 11, no. 12: 693. https://doi.org/10.3390/fermentation11120693

APA Style

Lima, A. B., de Almeida, K. L., Lima, B. E. T. d., Haddi, K., Passetti, L. C. G., Rosado, G. L., & Bento, C. B. P. (2025). Evaluation of Plant Essential Oils as Natural Alternatives to Monensin in In Vitro Ruminal Fermentation. Fermentation, 11(12), 693. https://doi.org/10.3390/fermentation11120693

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