Human diets have shifted in recent decades, with consumers increasingly demanding products that meet environmental and health standards(Komarek et al., 2021). This awareness has intensified pressure to restrict the use of additives, particularly ionophore antibiotics such as monensin, which is widely used to modify rumen fermentation, maximize performance, and prevent nutritional disorders such as ruminal acidosis (Khattab et al., 2020).

Consequently, growing interest in natural alternatives to ionophore antibiotics has driven research over the past decade, with essential oils (EOs) emerging as promising feed additives (Taban et al., 2021); (Caroprese et al., 2023). These lipophilic, volatile compounds possess antibacterial, antifungal, antioxidant, and anti-inflammatory properties, highlighting their potential as natural feed additives in animal nutrition (Taban et al., 2021). Among them, copaiba (Copaifera sp.) is a common name for various species distributed across Brazil’s Amazon and Central-West regions. Copaiba essential oil is extracted from different Copaifera species (Frazão et al., 2023). Phytochemical studies have shown that copaiba oils are composed of a mixture of sesquiterpenes and diterpenes, with copalic acid, β-caryophyllene, and α-copaene being the primary components (Frazão et al., 2023). Copaiba essential oil exhibits pronounced antimicrobial activity against a range of gram-positive bacteria, inducing cell wall disruption and the consequent leakage of cytoplasmic constituents (Rodrigues et al., 2020). In ruminants, suppression of gram-positive populations can favor the proliferation of gram-negative bacteria, thereby enhancing propionate synthesis in the rumen and improving overall animal performance (Moura et al., 2017). Thyme (Thymus vulgaris) essential oil is primarily characterized by its active compound, thymol, which exhibits broad-spectrum antimicrobial properties by altering membrane permeability in both gram-positive and gram-negative bacteria (Shaaban et al., 2021). According to (Shaaban et al., 2021), thymol supplementation modulates rumen fermentation by increasing volatile fatty acid (VFA) production while decreasing ruminal ammonia-N concentration.

Although copaiba and thyme essential oils have been evaluated individually in ruminants, to our knowledge, no study has directly compared both oils against monensin in the same in vivo sheep trial while integrating ruminal fermentation and methane responses with ingestive behavior, nitrogen metabolism, and infrared thermography. In this context, we hypothesized that copaiba and/or thyme essential oils can improve ruminal fermentation and reduce methane emissions, potentially matching or surpassing the effects of monensin in sheep. Therefore, this study aimed to evaluate the impact of these essential oils, compared with monensin, on nutrient intake, digestibility, nitrogen metabolism, ingestive behavior, infrared thermography, ruminal parameters, and estimated methane production.

There was an interaction between treatment and experimental period (p<0.05) for DM, CP, and CF intake (Table 2). The inclusion of additives (MON, CO, and TM) in the sheep’s diet increased dry matter intake (p<0.001) (Table 2). The TM increased CP intake, whereas MON decreased it (p=0.02). The CF intake showed a similar pattern (p = 0.02), mirroring the positive effect of TM on CP intake. The other variables were not influenced by the addition of the additives (p≥0.05) (Table 2). Regarding nutrient digestibility, only the digestibility coefficient of crude protein (dcCP) was influenced by treatment (p=0.03). Specifically, CO increased dcCP by approximately 14.46% compared to the control group (CON) (Table 2).

There was no interaction between treatment and experimental period (p≥0.05) for urinary excretion of purine derivatives and microbial efficiency (Table 3). The additives increased DOMR (p=0.05) compared to CON, with MON and TM being more efficient (Table 3). Similar patterns were observed in microbial nitrogen (MN) and microbial protein synthesis (p<0.05). However, the additives increased Uae (p=0.04) compared to CON (Table 3).

There was only an interaction between treatment and experimental period in the NI (p=0.03) (Table 4). The CO showed better NB (p=0.01) than the other treatments (Table 4). Although MON had lower NI than TM (p=0.03), urinary nitrogen loss was 0.23 g day-1 smaller in CO than in MON (p=0.04) (Table 4).

When evaluating ingestive behavior, we did not observe any interaction between treatment and period (p≥0.05) (Table 5). However, Rum was affected by treatments (p=0.01), with TM-treated sheep exhibiting the longest rumination duration, whereas MON resulted in the shortest rumination period (Table 5). No differences (p≥0.05) were observed in the other behavioral parameters (Table 5).

There was no interaction between treatment and period (p≥0.05) on infrared thermography (Table 6). MON reduced rumen temperature (p=0.04); however, although it also reduced the cannon region temperature (p<0.001), the essential oils CO and TM also reduced cannon temperature (Table 6).

The additives influenced (p<0.05) the ruminal parameters (Figure 1). MON prevented the reduction in pH (p=0.002) during the post-feeding hours, whereas CO and TM were not effective in preventing the pH drop (Figure 1a), which increased the redox potential (p=0.012) (Figure 1b). NH3-N content increased (p=0.002) with the use of CO (Figure 1c). Regarding VFA, the additives did not influence propionic (p=0.368) and butyric (p=0.709) acids. However, MON reduced acetic acid concentrations (p<0.001) and, consequently, estimated methane production (p<0.001) (Figures 1d and e). Despite this, CO also reduced methane emissions compared to TM and CON (Figures 1d and e).

The increased DMI in sheep resulting from the additives can be explained by the mechanisms of these compounds. Although the literature indicates that MON and CO typically improve feed efficiency by shifting fermentation toward propionate production, a more energetically efficient pathway for ruminants (Zhang et al., 2022), this specific VFA shift was not observed in the present study. MON primarily affects DMI by modulating ruminal fermentation, enhancing digestion and nutrient absorption, and stimulating feed intake (Luo et al., 2024). In contrast, EOs act more broadly by promoting changes in the ruminal microbiota and possessing appetite-stimulating properties (Moura et al., 2017); (Luo et al., 2024). (Shaaban et al., 2021), who also investigated the effects of TM in sheep, noted that the presence of bioactive compounds, along with their pleasant aroma and flavor, can stimulate feed intake by increasing saliva secretion. Additionally, the TM was observed to enhance feed passage rate, thereby increasing DMI. However, the increased CP intake may be supported by improved nitrogen metabolism. As previously mentioned, EOs can inhibit certain Gram-positive bacteria, including hyper-ammonia-producing species such as Clostridium sticklandii and Peptostreptococcus anaerobius(Dorantes-Iturbide et al., 2022), as well as ruminal protozoa (Orzuna-Orzuna et al., 2022). This modulation may reduce ruminal proteolysis and amino acid deamination, thereby decreasing ammonia-N production. As a result, less protein is degraded in the rumen, allowing a larger portion of dietary protein to bypass ruminal fermentation and be absorbed in the small intestine as amino acids (Cavallini et al., 2025). EOs may enhance nutrient digestibility by modulating rumen microbial populations and enzymatic activity (Besharati et al., 2021). This fact is supported by the higher dCP values (Table 2) observed with EO supplementation, along with an increase in NB (Table 4), indicating greater nitrogen retention, particularly in response to CO, followed by TM. However, the reasons for the increase in CF intake remain unclear.

The use of MON and EOs can modify ruminal fermentation, affect the ruminal microbiota, and improve nutrient utilization, thereby benefiting both DOMR and EMPS and ultimately improving MN (Table 3)(Susanto et al., 2023). Monensin has been reported to promote increased propionate production and reduce energy loss through methane emissions (Tseten et al., 2022). Although no increase in propionate concentration was observed in the present study, improvements in DOMR, EMPS, and methane mitigation suggest that MON enhanced ruminal fermentation efficiency through mechanisms beyond measurable changes in ruminal propionate concentration. EMPS is directly related to the effectiveness of microorganisms in utilizing nitrogen. Similarly, EOs are effective in microbial protein synthesis. Like MON, they reduce methane production and improve fiber digestibility, increasing the availability of nutrients for ruminal microorganisms and optimizing EMPS (Króliczewska et al., 2023). The ability of MON and TM to reduce NH₃-N concentrations (Figure 1c) is particularly relevant, as it indicates the inhibition of proteolytic bacteria, thereby preventing excessive protein degradation and nitrogen loss as ammonia(Wang et al., 2024). Improving nitrogen utilization is crucial because, according to (Hartinger et al., 2018), high ruminal ammonia levels often lead to increased nitrogen excretion rather than its incorporation into microbial protein. Additives decreased NU relative to the CON; however, CO was the most effective in improving NB (Table 4), indicating enhanced nitrogen retention. CO demonstrated the highest NB values among the treatments, suggesting superior nitrogen conservation. This result could be attributed to CO’s selective targeting of Gram-positive bacteria, which are implicated in excessive proteolysis, thereby reducing nitrogen losses (Moura et al., 2017). The lower NU in additive-treated sheep suggests that more nitrogen was directed towards microbial protein synthesis or retained in body tissues (Hartinger et al., 2018), enhancing overall nitrogen efficiency.

he increase in rumination time observed in TM-treated sheep (Table 5) is likely associated with their high DMI (Table 2). Prolonged rumination enhances saliva production, which provides buffering compounds such as bicarbonate and phosphate, contributing to the stability of ruminal pH (Mao & Wang, 2025). In contrast, the shortest rumination time recorded for MON-treated sheep (Table 5) is consistent with its well-established ability to shift ruminal fermentation towards higher propionate production (Russell & Houlihan, 2003), although this response was not detected in the present study. Since propionate is a more energy-efficient volatile fatty acid that requires less fermentation and chewing effort (Russell & Houlihan, 2003), the reduced need for the physical breakdown of fibrous material may account for the shorter rumination period. Despite this decrease in rumination time, MON-treated sheep maintained a stable ruminal pH (Figure 1a), likely due to monensin’s inhibitory effects on lactate-producing bacteria, thereby helping prevent acid accumulation (Russell & Houlihan, 2003).

The infrared thermography results further elucidate the metabolic effects of these additives, particularly MON. The lower rumen and paw temperatures observed in MON-treated sheep (Table 6) suggest reduced metabolic heat production. This observation aligns with MON’s known role in modifying fermentation to decrease methane and acetate production (Figures 1d and e) while favoring propionate synthesis, which generates less heat (Onan-Martinez et al., 2025). Although propionate concentration was not significantly affected in the present study, the observed changes are consistent with improved fermentation efficiency reported for MON. Reduced heat production can be particularly beneficial in warm climates, where excessive metabolic heat can contribute to heat stress and negatively impact feed intake (Kim et al., 2022). Additionally, the lower paw temperature in MON-treated sheep indicates improved thermoregulation, potentially through enhanced circulatory efficiency and reduced heat-dissipation requirements (Kim et al., 2022). The OEs showed intermediate results, highlighting broader physiological benefits beyond fermentation modulation and demonstrating their potential as a strategic dietary additive for reducing heat stress in ruminants.

In terms of ruminal parameters, the increased ruminal pH and redox potential (E_h) with MON supplementation (Figures 1a and b) align with its previously mentioned ability to inhibit gram-positive bacteria and modulate ruminal fermentation, thereby reducing acid accumulation ((Onan-Martinez et al., 2025). The EOs resulted in slightly lower ruminal pH and Eh than MON and CON (Figures 1a and b). However, the pH remained within the normal range of approximately 5.8 to 6.8 (Hungate, 2013); (Mao & Wang, 2025), and Eh was +100 mV or less and less than −250 mV(Huang et al., 2018). This result indicates that the EOs did not cause excessive acidification of the rumen environment, suggesting that their antimicrobial activity did not significantly disrupt the balance between acid-producing and acid-utilizing microorganisms, nor did it alter the electron transfer balance in the rumen. Estimated methane production (Figure 1e) followed a pattern similar to that of acetic acid concentration (Figure 1d), as expected, since acetate is primarily synthesized by Gram-positive bacteria and the associated hydrogen is utilized by Methanogenic archaea to produce methane (Hungate, 2013). MON and CO exhibited the lowest levels of methane and acetic acid production during the initial hours, likely due to their similar mechanisms of action (Moura et al., 2017). This has been reported to inhibit methanogenic microorganisms and, in many studies, shift fermentation pathways toward propionate production (Moura et al., 2017). However, in the present study, methane and acetate were reduced, with no significant change in propionate concentration. This outcome is particularly beneficial, as estimated methane production accounts for approximately 6% to 18% of the diet’s gross energy loss during fermentation (Min et al., 2022).

While the additives increased dry matter intake, CO and TM maximized crude protein intake and digestibility. EOs enhanced nitrogen availability for microbial protein synthesis, thereby improving nitrogen balance. In contrast, MON reduced ruminal and cannon region temperatures, likely reflecting changes in ruminal fermentation and lower heat production. Both MON and CO reduced the acetate concentration, thereby lowering methane production. Overall, EOs were as effective as MON in reducing estimated methane production while improving nutrient utilization and nitrogen use efficiency in sheep.

We certify that there is no conflict of interest with any financial, personal, or other relationships with other people or organizations related to the material discussed in the manuscript.

This study was funded by the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro - FAPERJ (RJ, Brazil), process number E-26/204.325/2025, Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq, and Universidade Estadual do Norte Fluminense under Grant PAPIC SEI-260002/006970/2024.

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