Discussion

The present study investigated the chemical composition of P. abrotanoides essential oil and its effects on in vitro rumen fermentation parameters, methane production, protozoa population, nutrient degradability and microbial efficiency indices. The results demonstrated that the essential oil, rich in oxygenated monoterpenes (mainly 1,8‐cineole and camphor), significantly reduced methane production and protozoa population while increasing microbial biomass synthesis efficiency and PF, albeit with some adverse effects on fibre degradability at higher doses. These findings support the hypothesis that P. abrotanoides essential oil, through its bioactive antimicrobial constituents, can establish a balance between methane reduction and maintenance of rumen fermentation efficiency at an appropriate dosage level.

Chemical Composition of Perovskia abrotanoides Essential Oil

The GC–MS analysis of the essential oil (Table 2) revealed 33 compounds, with 1,8‐cineole (16.10%), camphor (13.80%), α‐pinene (8.40%), δ‐3‐carene (6.81%) and (E)‐caryophyllene (4.21%) as the major constituents. The essential oil yield was 1.5% (w/w) based on dry plant weight. This yield is comparable to previously reported values for P. abrotanoides from different regions of Iran. Shahraki et al. (2013) reported essential oil yields of 1.8% and 1.6% for samples collected from Kiasar (Mazandaran) and Golestan National Park, respectively. Similarly, Pourhosseini et al. (2018) observed yields ranging from 0.7% in stalks to 2.3% in flowers, with leaf yield of 1.0%. Ghaffari et al. (2018) analysed 17 populations across Iran and reported a broader yield range of 1.25%–3.61%, with the highest yield (3.61%) recorded in a Semnan population and the lowest (1.25%) in an Isfahan population (Keshe). The yield obtained in the present study (1.5%) falls well within these reported ranges, confirming that P. abrotanoides essential oil yield is influenced by geographical origin, climatic conditions and plant part analysed, while remaining consistently moderate across different regions.

These findings are consistent with previous reports on P. abrotanoides essential oil composition from various regions of Iran. Morteza‐Semnani (2004) reported camphor (34.1%), 1,8‐cineole (18%) and α‐pinene (6.5%) as the main components. Similarly, Mahboubi and Kazempour (2009) identified camphor (23%), 1,8‐cineole (22%) and α‐pinene (12%) in their sample from Kashan, Iran. Ghaffari et al. (2018) analysed 17 populations of P. abrotanoides from different Iranian provinces and found camphor (4.05%–35.94%), 1,8‐cineole (7.15%–24.34%), borneol (0%–21.75%) and α‐pinene (2.05%–10.33%) as the main constituents. Our results fall within these reported ranges, confirming that P. abrotanoides essential oil is characterized by high levels of oxygenated monoterpenes, particularly 1,8‐cineole and camphor.

However, considerable variation exists in the chemical composition of P. abrotanoides essential oil depending on geographical origin, climatic conditions, altitude, soil properties and plant part analysed. Pourhosseini et al. (2018) reported that flower essential oil contained higher amounts of camphor (18.8%) and 1,8‐cineole (16.5%) compared to leaves (10.1% and 11.4%, respectively) and stalks (21.0% and 16.3%, respectively). Shahraki et al. (2013) also found significant differences between two natural habitats in Golestan and Mazandaran provinces, with the Kiasar region (Mazandaran) showing higher essential oil yield (1.8%) and a different major compound profile (higher 1,8‐cineole and camphor) compared to Golestan National Park (1.6%). In contrast, one study from the same Kiasar region reported α‐terpineol (21.9%–32.0%) and n‐octanol (17.4%–23.3%) as the dominant compounds (Kolbady Nejad et al. 2013), indicating the existence of different chemotypes within the species. Ashraf et al. (2014) reported an entirely different profile from Pakistan, with (E)‐9‐dodecenal (66.5%) as the major component in stem essential oil, highlighting the profound impact of geographical and genetic factors on essential oil composition. These variations are attributable to differences in genotype, agronomic practices, climatological factors, developmental stage, post‐harvest storage and extraction methods (Ashraf et al. 2014).

Effect on Methane Production and Gas Production Parameters

Increasing levels of P. abrotanoides essential oil reduced methane production by approximately 57.5%. This reduction is most likely attributable to the high concentration of oxygenated monoterpenes, particularly 1,8‐cineole and camphor, which are known to suppress methanogenic archaea directly and indirectly through inhibition of ruminal protozoa. The lipophilic nature of these compounds disrupts microbial cell membranes and alters hydrogen metabolism, thereby limiting the substrate available for methanogenesis. These findings are consistent with previous studies demonstrating the antimethanogenic potential of essential oils rich in oxygenated monoterpenes (Cobellis et al. 2016; Nunes et al. 2023; Kelly and Kebreab 2023).

Patra and Yu (2012) evaluated five essential oils (clove, eucalyptus, garlic, origanum and peppermint) at doses up to 1.0 g/L and reported methane reductions ranging from 17.6% (eucalyptus) to 87% (origanum). Although our P. abrotanoides essential oil contains 1,8‐cineole (a major component of eucalyptus oil), its methane reduction efficacy (57.5% at 500 mg/L) was considerably higher than that reported for pure eucalyptus oil (17.6% at 1.0 g/L) by Patra and Yu (2012). This enhanced efficacy may be attributed to the synergistic effects of multiple bioactive compounds in P. abrotanoides essential oil, including camphor, α‐pinene and δ‐3‐carene, in addition to 1,8‐cineole.

Benetel et al. (2022) tested 10 essential oils in an in vitro rumen fermentation system and found that oregano and white thyme essential oils caused drastic reductions in total gas production (up to 75%) and methane production, with net methane values dropping from 6.92 mL (control) to 0.17 mL (oregano) and 0.57 mL (white thyme) at 500 mg/L. These reductions were attributed to the high content of thymol and carvacrol, phenolic monoterpenes with strong antimicrobial activity. Although our essential oil does not contain thymol or carvacrol, its high content of 1,8‐cineole and camphor appears to confer substantial antimethanogenic activity, albeit less potent than oregano oil.

The mechanism by which essential oils reduce methane production is primarily through direct inhibition of methanogenic archaea and/or indirect suppression of protozoa, which are symbiotically associated with methanogens (Patra and Yu 2012). In the present study, total protozoa population decreased linearly (p < 0.0001) from 5.17 × 105 cells/mL in the control to 3.42 × 105 cells/mL at 500 mg/L (Table 4). This antiprotozoal effect likely contributed to the observed reduction in methane production, as protozoa provide hydrogen for methanogens. Patra and Yu (2012) similarly reported that all tested essential oils reduced protozoal abundance, with origanum and peppermint oils causing the greatest reductions (nearly 3 log units). The antiprotozoal activity of essential oils is attributed to their lipophilic nature, which allows them to penetrate the protozoal cell membrane and disrupt cellular functions (Patra and Yu 2012).

Effect on Protozoa Population and Fermentation Parameters

Total protozoa population decreased linearly (p < 0.0001) with increasing essential oil levels, from 5.17 × 105 cells/mL in the control to 3.42 × 105 cells/mL at 500 mg/L (Table 4). This marked reduction is likely attributable to the antimicrobial activity of the major oxygenated monoterpenes present in the essential oil, particularly 1,8‐cineole and camphor. Owing to their lipophilic nature, these compounds can penetrate protozoal cell membranes, disrupt membrane integrity, increase permeability and interfere with essential metabolic processes, ultimately reducing protozoal survival and activity in the rumen ecosystem. This finding is consistent with Mahboubi and Kazempour (2009), who demonstrated that P. abrotanoides essential oil and its principal constituents (camphor and α‐pinene) possess strong antimicrobial activity. Although protozoa were not evaluated in their study, the proposed mechanism of membrane disruption agrees with previous reports describing the mode of action of essential oils against rumen microorganisms (Patra and Yu 2012). The reduction in protozoal abundance may also partially explain the lower methane production observed in the present study because ruminal protozoa maintain a close symbiotic association with methanogenic archaea and contribute substantially to hydrogen transfer required for methanogenesis.

Total VFA concentration decreased linearly (p < 0.0001) from 96.00 mmol/L in the control to 79.25 mmol/L at 500 mg/L (Table 4). This decrease most likely reflects a reduction in overall microbial fermentation activity resulting from partial inhibition of fermentative microorganisms, particularly cellulolytic bacteria responsible for carbohydrate degradation. Lower fermentation activity inevitably reduces the production of fermentation end‐products, including VFA. This observation agrees with Patra and Yu (2012), who reported decreased total VFA concentrations following supplementation with clove and oregano essential oils, whereas garlic, eucalyptus and peppermint oils produced little or no effect. Although reduced VFA production may indicate some depression of fermentative activity at higher doses, it also suggests that the antimethanogenic effect of the essential oil is accompanied by a shift in rumen fermentation pathways rather than complete inhibition of microbial metabolism.

The NH3–N concentration decreased linearly (p < 0.0001) from 24.25 mg/dL in the control to 17.12 mg/dL at 500 mg/L (Table 4). The reduction in ammonia concentration is likely associated with inhibition of hyper‐ammonia‐producing bacteria, thereby reducing amino acid deamination and improving nitrogen retention within the microbial ecosystem. Such an effect may increase the availability of nitrogen for microbial protein synthesis instead of ammonia accumulation. Patra and Yu (2012) similarly reported lower ammonia concentrations after supplementation with clove and oregano essential oils, attributing this response to reduced deamination activity. Mahboubi and Kazempour (2009) also demonstrated the antimicrobial activity of camphor and α‐pinene, suggesting that these compounds may inhibit ammonia‐producing microorganisms. Importantly, NH3–N concentrations remained above the minimum threshold required for optimal microbial growth (Satter and Slyter 1974), indicating that nitrogen availability for microbial protein synthesis was not compromised despite the reduction in ammonia concentration.

In contrast, pH increased linearly (p < 0.0001) from 6.63 in the control to 6.85 at 500 mg/L (Table 4). The increase in ruminal pH is a logical consequence of reduced fermentation intensity and lower VFA production, as fewer organic acids accumulated in the incubation medium. Maintaining ruminal pH within this range may also contribute to a more stable rumen environment by reducing the risk of excessive acidification. Similar increases in ruminal pH following essential oil supplementation have been reported by Patra and Yu (2012). Therefore, the observed increase in pH appears to represent an indirect response to changes in fermentation activity rather than a direct effect of the essential oil itself.

Effect on Nutrient Degradability and Microbial Efficiency

Increasing levels of essential oil linearly decreased (p < 0.0001) NDF degradability (from 50.25% to 40.50%), OM degradability (from 75.01% to 64.50%) and DM degradability (from 73.75% to 65.00%) (Table 5). The reduction in nutrient degradability is likely associated with inhibition of fibrolytic microorganisms responsible for structural carbohydrate degradation. Oxygenated monoterpenes, such as 1,8‐cineole and camphor, may suppress the growth and enzymatic activity of cellulolytic bacteria, thereby limiting fibre digestion and reducing substrate degradation. This inhibitory effect becomes more pronounced at higher essential oil concentrations because antimicrobial activity extends beyond methanogens to beneficial fibrolytic microorganisms. These findings agree with Patra and Yu (2012), who reported reductions in apparent DM and NDF degradability following supplementation with several essential oils. Likewise, Benetel et al. (2022) observed decreased in vitro DM digestibility with white thyme essential oil. Therefore, although the essential oil effectively mitigated methane production, excessive supplementation may compromise nutrient utilization, highlighting the importance of identifying an optimal inclusion level that balances environmental benefits with digestive efficiency.

Despite the reduction in fibre degradability, microbial biomass production increased linearly (p < 0.0001) from 58.32 mg in the control to 84.63 mg at 500 mg/L (Table 5). Similarly, microbial biomass synthesis efficiency increased from 30.87% to 55.46%, and the PF increased from 3.18 to 4.94 mg truly degraded OM/mL gas produced. These responses indicate that microbial metabolism became more efficient, allowing a greater proportion of degraded substrate to be incorporated into microbial biomass rather than being lost as gaseous fermentation products. Inhibition of methanogenesis reduces energy losses and may redirect reducing equivalents towards alternative metabolic pathways and microbial growth. Consequently, the remaining microbial community may utilize available nutrients more efficiently despite lower overall substrate degradation. Similar improvements in PF following essential oil supplementation have been reported by Benetel et al. (2022), who suggested that increased PF reflects enhanced microbial efficiency. Patra and Yu (2012) also proposed that inhibition of methanogenesis may alter hydrogen utilization pathways and promote the growth of alternative microbial groups. Collectively, these findings suggest that P. abrotanoides essential oil has the potential to improve microbial energy utilization efficiency, although this beneficial effect should be balanced against the observed reduction in fibre degradability when determining the optimal supplementation level.