Nutritional value of Thymus algeriensis (Boiss. & Reut.) and the impact of its essential oil on in vitro digestibility

Abaidia Abdelghafour^1,2, Laadjal Abdessatar^3,4, Selmi Houcine⁵ and Zeraib Azzeddine^1,6

¹ Department of Agronomy, Faculty of Nature and Life Sciences, University of Abbes Laghrour, PB 1252 road of Batna, Khenchela, 40004, Algeria
laadjal-abdessatar@univ-eltarf.dz
² Laboratory of Epidemiological Surveillance, Health, Production and Reproduction, Experimentation and Cell Therapy of Domestic and Wild Animals, Chadli Bendjedid University, PB 73, 36000, El-Tarf, Algeria
³ Agropastoralism Research Center (CRAPast ), 17000 Djelfa, Algeria
⁴ Laboratory of Agriculture and Ecosystem Functioning, Chadli Bendjedid University, PB 73, 36000, El-Tarf, Algeria
⁵ Laboratory of Sylvo‑Pastoral Resources, Sylvo‑Pastoral Institute of Tabarka, University of Jendouba, Jendouba, Tunisia
⁶ Laboratory of Genetics, biotechnology and valorization of bio-resources, University of Biskra, Algeria

Abstract

This study aimed to characterize the nutritional composition of Thymus algeriensis and to evaluate the effect of its essential oil (EOTa) on in vitroruminal fermentation and methane production. Chemical analysis revealed that T. algeriensis contains acceptable levels of crude protein (9.9% DM), high fiber contents (58% DM NDF, 43% DM ADF and 18% DM ADL) and moderate concentrations of phenolic compounds (30 and 16 g/kg DM for total phenols and total tannins, respectively). Increasing doses of EOTa (0, 10, 20, 40, 60 and 80 μl) were tested with vetch–oat hay using the gas production technique. Supplementation with 10 μl of EOTa enhanced organic matter digestibility, metabolizable energy and volatile fatty acid production, whereas doses higher than 20 μl significantly reduced total gas and methane production. Overall, the results highlight the nutritional value of T. algeriensis and suggest that its essential oil may beneficially modulate ruminal fermentation while mitigating methane emissions. However, further investigations, particularly in vivo studies, are required to confirm these findings.

Keywords: chemical composition, methane mitigation, phenolic compounds, rumen fermentation

Introduction

Nutrition represents a key lever for improving both the productivity and sustainability of ruminant production systems. Optimizing forage digestibility and nutrient utilization efficiency is essential to reducing production costs while enhancing animal performance (Caroprese et al 2023 ; Susanto et al 2023). In this context, the use of natural additives derived from aromatic plants has gained increasing attention, driven by the gradual restriction of certain synthetic additives and the growing demand for sustainable alternatives (Kholif and Olafadehan 2021).

Among these natural additives, essential oils (EOs) stand out for their potential to modulate ruminal fermentation, influence volatile fatty acid (VFA) production and improve feed efficiency, while in some cases mitigating methane emissions (Belanche et al 2020 ; Joch et al 2019 ; Silvestre et al 2023). However, their effectiveness varies depending on plant species, chemical composition and administered dose (Garcia et al 2020 ; Torres et al 2020).

Thymus algeriensis (Boiss. & Reut.) is an endemic species of the semi-arid regions of North Africa (Tunisia, Algeria, Libya), known for its richness in essential oils dominated by thymol and carvacrol. Although traditionally used for its aromatic and medicinal properties, its zootechnical potential remains poorly investigated. Nevertheless, previous studies reported that dietary inclusion of thyme oil (0.1–0.2% of dry matter) in growing lambs improved feed efficiency without adverse effects (El-Naggar et al 2017).

We hypothesized that the essential oil of T. algeriensis (EOTa) could enhance ruminal fermentation and digestibility while modulating methane production. Therefore, the objectives of this study were (i) to characterize the nutritional composition of T. algeriensis and (ii) to evaluate, in vitro, the effect of different doses of its essential oil on vetch–oat fermentation and methane emissions.

Materials and methods

Plant material

The aerial parts (leaves and young shoots) of thyme (Thymus algeriensis) were harvested in the commune of Ain Mimoun, Khenchela province (Northeastern Algeria), an agro-silvopastoral zone located at 35.284°N and 6.596°E in a semi-arid bioclimatic stage characterized by cold winters and hot, dry summers. Sampling was conducted during the autumn season of 2021. Plant material was oven-dried for 72 h at 45°C to avoid degradation of phenolic compounds, ground to pass through a 1 mm sieve and stored in airtight containers until further analysis.

Photo 1. Thyme plant (Thymus algeriensis) (Fakchich et al 2024)

Essential oil extraction

Essential oil was extracted by steam distillation. Fresh plant material was placed on a grid above the water level in the distillation unit. As the water was heated, steam passed through the plant material, enriching it with volatile constituents. The vapor was condensed using a cooling system and the distillate collected in a glass flask. The hydrosol was separated from the essential oil, which was recovered in dark glass vials. Distillation was carried out for 4 h at 100°C.

Primary and secondary chemical composition

Samples of T. algeriensis and vetch-oat hay were analyzed for dry matter (DM) and ash according to AOAC (1990). Crude protein (CP) was determined by the Kjeldahl method (ISO 2009). Cell wall fraction-neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL)-were determined following the procedure of Van Soest et al (1991).

Total phenols (TP) were quantified using the Folin-Ciocalteu reagent and total tannins (TT) were estimated as the difference between total phenols measured before and after treatment with polyvinylpolypyrrolidone (PVPP), following Makkar (2003). Results were expressed as g tannic acid equivalent (TAE) per kg DM.

In vitro and gas production kinetics

Rumen contents were collected from local cows grazing forest pastures. The contents were immediately filtered through four layers of surgical gauze and the rumen fluid was stored in a pre-warmed thermos at 39°C for laboratory use.

Gas production was measured following the technique of Menke (1988). Briefly, 0.3 g of substrate (T. algeriensis or vetch-oat hay) was incubated with 30 ml of inoculum (20 ml of artificial saliva and 10 ml of rumen fluid) in calibrated 100-ml glass syringes, which were placed in a water bath at 39°C for the entire incubation period. Each treatment was run in duplicate, with two blanks (inoculum only) included as controls. For the essential oil experiment, vetch-oat hay was supplemented with essential oil of T. algeriensis (EOTa) at doses of 10, 20, 40, 60 and 80 μl. Each treatment was incubated in duplicate, with corresponding blanks.

Gas volume was recorded at 2, 4, 6, 8, 12, 24, 48 and 72 h of incubation. After each incubation, 4 ml of NaOH (10 N) was injected into the syringes to absorb CO₂ and piston retraction was used to quantify methane production.

Gas production kinetics and in vitro digestibility parameters

Gas production kinetics were estimated by nonlinear regression using the ‘nlme’ package in R (version 4.4.1), following the exponential model proposed by Ørskov & Mc Donald (1979) :

Y = a + b (1 – e-ct)

where :

Y : total gas volume produced (ml),

a : gas volume from the soluble, rapidly fermentable fraction (ml),

b : gas volume from the insoluble but potentially fermentable fraction (ml),

c : gas production rate(h-1),

t : incubation time (h).

Organic matter digestibility (OMD) were estimated from 24 h gas production using equation proposed by Menke (1988) and metabolizable energy (ME) with volatile fatty acid (VFA) concentrations according Getachew et al (1998) :

OMD (%) = 14,88 + 0,889 GP + 0,45 CP + 0,0651 Ash

EM (MJ/kg DM) = 2,2 + 0,1357 GP + 0,0057 CP + 0,000859 CP

VFA (mmol/syringe) = 0,0239 GP-0,0601

where : GP = gas volume (ml/0.3g DM) after 24 h incubation.

Statistical analysis

The effects of essential oil dose on in vitrofermentation parameters were analyzed by analysis of variance (ANOVA) using the GLM procedure of Minitab 17. Means were compared by Tukey’s multiple range test.

The statistical model was:

Yij = µ + Ai + Eij

where :

Yij : observed parameter,

µ : overall mean,

Ai : effect of the ith dose,

Eij : residual error for the jth replicate.

Results and discussion

Nutritional characterization of Thymus algeriensis

Chemical composition

The chemical composition of T. algeriensis and vetch-oat hay (VOH) revealed significant differences between the two substrates (Table 1). VOH showed higher values for most parameters

(p< 0.05), except for ADF and ADL fractions, which were statistically similar (p> 0.05). Notably, T. algeriensis was characterized by a high content of phenolic compounds (30.19 ± 2.99 g TAE/kg DM for total phenols), which were absent in VOH.

The high dry matter (DM) content of T. algeriensis (84.77%) reflects the adaptation of this species to the semi-arid climate of the study area. This value is substantially higher than that reported by Jiang et al (2020) for the same species in China (40.89%), but lower than the 97.4% reported for T. serrulatus under Sahelian conditions (Eshete et al 2012). Such variability highlights the strong influence of edaphoclimatic conditions on the chemical composition of Mediterranean aromatic plants (Ammar et al 2005).

The mineral matter content (10.27% DM) is consistent with observations reported by Moujahed et al (2011) for Tunisian T. capitatus. The crude protein content (9.99% DM), comparable to the 9% DM reported by Jiang et al (2020), exceeds the minimum threshold of 7% required to sustain optimal rumen microbial activity (Van Soest 1994). This moderate protein level, together with the presence of secondary metabolites, suggests a promising potential for T. algeriensis as a dietary supplement rather than as a main forage resource.

The fiber fractions of T. algeriensis (NDF : 58.06% DM ; ADF : 43.17% DM) were higher than the values reported by Jiang et al (2020) for the same species (43 and 39% DM, respectively), suggesting a higher degree of lignification under the present experimental conditions. However, the ADL content (18.19% DM), although relatively high, remained below the critical values of 22–23% DM reported in other thyme species (Moujahed et al 2011 ; Parlak et al 2011), thereby preserving acceptable digestibility potential.

In vitro ruminal fermentation parameters

The fermentability assessment of T. algeriensis revealed a distinct fermentation profile compared with VOH (Table 2). Total gas production (47.76 ml/0.3 g DM) and potential gas production (a+b : 47.97 ml) were significantly lower than those of VOH (p< 0.05), whereas fermentation rates remained similar between substrates (c : 0.06 vs. 0.05 ml/h ; p > 0.05).

The negative value of parameter “a” (-3.55 ml) deserves particular attention. This phenomenon, frequently observed in substrates rich in antinutritional compounds, reflects an initial “lag phase” during which microbial adaptation predominates over gas production (France et al 2000). Such a delay may be explained by the need for the ruminal microbiota to synthesize specific enzymes or to develop detoxification mechanisms against phenolic compounds (Patra & Saxena 2010).

Gas production, as a direct indicator of fermentative activity, is positively correlated with volatile fatty acid (VFA) synthesis, the main energy substrates for ruminants (Kamalak et al 2011). The moderate value observed here (47.76 ml) suggests controlled fermentation, potentially advantageous for limiting energy losses as gas while maintaining adequate VFA production (Knapp et al 2014).

Digestibility parameters confirmed the moderate yet relevant nutritional potential of T. algeriensis. Organic matter digestibility (50.84%) and metabolizable energy (7.03 MJ/kg DM) indicate acceptable energy utilization, although lower than that of VOH. These values, consistent with the presence of phenolic compounds and a high lignin fraction, are nevertheless sufficient to support its use as a dietary supplement (Al-Dabisi et al 2023).

The most remarkable result concerns methane production, which was significantly reduced (12.41 vs. 36.29 ml/0.3 g DM ; p< 0.001). This 66% decrease compared with the control suggests a strong antimethanogenic effect, likely associated with the action of bioactive compounds on methanogenic archaea (Bodas et al 2012). This property is particularly relevant in the current context of mitigating greenhouse gas emissions from livestock production.

Effect of Thymus algeriensis essential oil on in vitro ruminal fermentation

The incorporation of T. algeriensis essential oil (EOTa) induced a complex dose-dependent modulation of ruminal fermentation (Figure 1, Table 4). A biphasic response was observed: stimulation at low doses (10 μl), with a 6% increase in gas production (64.68 vs. 61.03 ml), followed by progressive inhibition at higher doses, reaching up to 80% reduction at 80 μl.

Figure 1. Gas production kinetics at different doses of Thymus algeriensis essential oil

At 10 μl, the selective antimicrobial activity of EOTa may have favored fibrolytic bacteria by suppressing predatory protozoa, a phenomenon previously documented for thymol and carvacrol (Benchaar 2021). This is consistent with the increased “b” fraction (72.81 vs. 69.69 ml), indicating improved degradation of structural substrates. Beyond 20 μl, the progressive inhibition suggests a non-selective antimicrobial effect, impairing the whole microbial consortium. At these higher concentrations, the disruption of bacterial membrane integrity by phenolic compounds becomes detrimental (Macheboeuf et al 2008). Interestingly, the maintenance -or even increase- of the fermentation rate “c” at inhibitory doses could reflect microbial adaptation or shifts toward alternative fermentative pathways.

Digestibility and metabolic parameters confirmed the existence of a functional optimum between 10 and 20 μl (Table 5). At 10 μl, organic matter digestibility (63.52 vs. 60.67%), metabolizable energy (8.88 vs. 8.44 MJ/kg DM) and VFA production (1.08 vs. 1.01 mmol/syringe) improved compared with the control, suggesting enhanced fermentative efficiency without impairing microbial activity.

Although individual VFA molar proportions were not detailed, the observed patterns likely involved a shift in the acetate-to-propionate ratio, a common feature of thymol-rich essential oils (Baraz et al 2018). Such a metabolic adjustment, favoring propionate production, enhances energy efficiency while reducing methane formation, as propionate acts as an alternative hydrogen sink (Khattab et al 2020).

The most striking outcome was the dose-dependent reduction in methane emissions. At 10 μl, methane decreased by 50% (18.14 vs. 36.29 ml), striking a balance between antimethanogenic activity and preservation of ruminal function. This reduction can be attributed to synergistic mechanisms: direct inhibition of methanogens, VFA profile modulation toward hydrogen-consuming pathways and possibly reduced protozoal activity (Benetel et al 2022).

Importantly, the antimethanogenic effect persisted even at inhibitory doses (60–80 μl), suggesting greater sensitivity of methanogenic archaea compared with fermentative bacteria. Such selectivity, already reported for mediterranean essential oils (Djabri et al 2016), offers promising perspectives for the development of dietary additives with environmental benefits. Roy et al (2015) further demonstrated that this selective inhibition can maintain long-term methane reduction without compromising zootechnical performance, particularly when essential oils are combined with other nutritional strategies.

Conclusion

This study highlightes the nutritional relevance of Thymus algeriensis, characterized by a moderate protein content and a high concentration of phenolic compounds, which may modulate ruminal fermentation. The in vitro trials demonstrate that the inclusion of low doses of T. algeriensis essential oil (10 μl) enhanced organic matter digestibility, metabolizable energy and volatile fatty acid production. In contrast, higher doses progressively inhibited gas production, reflecting a non-selective antimicrobial effect. A particularly noteworthy finding was the significant reduction in methane emissions, confirming the potential of this essential oil as a natural additive with environmental benefits.

Overall, T. algeriensis and its essential oil emerge as promising alternatives for improving feed efficiency in ruminants while reducing their carbon footprint. However, the dose-dependent variability observed underscores the need to establish optimal thresholds of inclusion. Further studies, particularly in vivo, are essential to validate these findings and to assess their long-term effects on animal performance and the sustainability of livestock production systems.

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