4. Discussion
Although there is already research on some EO using the in vitro technique of gas production, our work investigated additional EO and variables that were not previously considered. The effects of EO tend to be influenced by their majority variable components (including diet composition), making it difficult to parse out their effect on ruminant nutrition [
7]. The possible synergic or antagonistic interactions are difficult to measure and interpret [
7].
A reduction of TGP in animals consuming diets with
Lippia turbinate and
Tagetes minuta was observed by Garcia et al. (2020) [
19]. Those authors observed a reduction in in vitro gas production, without affecting the digestibility of the substrates in intermediate doses. In our study, oregano and white thyme EO caused a drastic reduction in TGP of up to 75%, indicating that these substances have high antimicrobial activity in ruminal conditions. To explain this relationship, Lambert et al. (2001) [
20] stated that oregano EO has molecules of thymol and carvacrol, and that these would be most responsible for its antimicrobial effect. These same molecules were observed in the chemical characterization of oregano and thyme EO.
García-García et al. (2011) [
21] showed that thymol alone may not be as effective at reducing CH
4 as the combination between it and carvacrol. When testing the isolated effects of carvacrol or thymol on
Listeria innocua, they obtained stronger effects for the first compound, while the binary effect of carvacrol plus thymol was more effective in inactivating bacterial growth, evidencing the synergistic effect between the components.
Garcia et al. (2020) [
19] observed that increasing doses of EO had progressively inhibitory action in substrates digestibility, with almost total inhibition when using 300 µL/L. In contrast, Khorrami et al.’s (2015) [
22] in vivo study observed that thyme and cinnamon essential oils (500 mg/kg DM) did not decrease feed intake and nutrient digestion.
Several factors can influence the TGP and consequently the production of CH
4 by the animals through rumen fermentation [
19]. One of the most important factors in this process is the extent of feed degradation. According to Bueno et al. (2005) [
12], the in vitro gas production system estimates the degradation of the feed (substrate). Previous studies also suggest that in vitro gas production assays should be complemented with residue determination at the end of the incubation process, since the gas measurements alone provide an incomplete explanation. The determination of the final residue indicates the amount of substrate that was effectively used during the in vitro fermentation process (Getachew et al., 1998) [
23].
Castilejos et al. (2006) [
24] and Fraser et al. (2007) [
25], when testing 500 ppm thymol (the primary compound of thyme EO) and cinnamaldehyde, also observed a reduction in the disappearance of IVDMD with these substances, as we also found. Other studies using EO showed a reduction in feed degradation [
26,
27], which would be a disadvantage for animal production in the use of this additive.
The search for EO that reduce ruminal CH
4 emissions without affecting feed degradation is important to improve feed efficiency and thus contribute to innovation in green technologies [
19]. Feeds with low degradation may make it difficult for rumen microorganisms to extract substrates and, consequently, result in nutrient limitation [
28].
In our study, some EO, such as citronella, ho wood, oregano and thyme, likely induced hydrophobic characteristics on the soluble portions of the feed substrate, preventing microbial attack and its metabolism. This was evidenced when analyzing the TGP drop and the maintenance in the degradability of the substrate with increasing inclusion levels.
The partition factors for both DM and OM in oregano and white thyme EO in our research were superior to the other EO when evaluated at a dosage of 500 mg/L. This may be explained by the low production of gases with a concurrent lower substrate disappearance in the presence of white thyme EO and constant substrate disappearance when oregano was added.
The quantification of ammonia concentration is an important indication of nitrogen use efficiency during in vitro bioassays, since 60% to 80% of the nitrogen incorporated by rumen microorganisms comes from ammonia [
29]. Its concentration may be affected both by the degradability content of CP present in the substrate and by rumen microorganism use. Feed CP functions as a source of rumen NH
3-N for ruminal microorganisms so it is essential to have sufficient energy available. Thus, rumen NH
3-N is a direct result of the quantity generated and the quantity used by rumen microorganisms [
30].
All the EO evaluated presented little variation in NH
3-N content; however, the EO of citronella and ho wood had a positive linear relation between NH
3-N and inclusion levels, suggesting a stabilization of the microbiota environment. The concentrations were always sufficient to support microbiota growth, all being well above the minimum value reported by Satter and Slyter (1974) [
31], of 5 mL/dL. In addition, they were above 13 mg/dL—the minimum value necessary to avoid compromising the availability of N for microorganisms, and the ingestion and digestibility of fiber [
28].
Benchaar et al. (2007) [
32] did not observe an effect of cinnamon (400 mg/L), oregano (200 mg/L) and thymol (200 mg/L) EO on rumen NH
3-N concentration in relation to the treatment without additives. Cardozo et al. (2005) [
33], when testing 0.3 mg/L of garlic, cinnamon and oregano EO, also did not observe a difference compared to the control diets without EO. However, garlic EO doses of 30 and 300 mg/L reduced ammonia concentration by 32.7% and 55.4%, for cinnamon EO by 31.6% and 61.9%, and for oregano EO by 26.9% and 64.5%, respectively. According to those authors, either EO decreased the deamination or microorganisms used the peptides and amino acids as a source of nitrogen, with a consequent reduction in NH
3-N.
More important than analyzing the effect of each EO on each variable related to ruminal fermentation is to analyze the influence and consequences that each exerts on the other [
34]. The absence of effects on total concentrations of SCFA can also be seen as a positive if it is accompanied by increased feed degradability as well as decreases in C
2:C
3 ratios and the production of CH
4.
A secondary metabolite can have a beneficial effect against methane-producing microorganisms but can also reduce the total concentration of SCFA and/or the degradability of feed in ruminants [
35]. There is an inverse relationship between C
3 and CH
4 production; the metabolic route of C
3 production, besides serving as a drain of H
+, still generates less of this ion compared to the C
2 or C
4 route [
28,
36]. By increasing the C
3 production, there is more competition with methanogenic archaea for substrate, generating less CH
4 production.
Citronella, globulus, staigeriana, ginger, melaleuca, oregano and white thyme OE had a positive linear relationship with C2:C3 ratios, indicating that the rumen was less energy efficient. However, the C2:C3 ratio and the maintenance of the degradability of coast-cross hay suggests that the addition of EO led to a decline in selectivity in cellulolytic bacteria, responsible for the degradation of fibrous substrates and the main producers of C2.
Diets rich in concentrate are more prone to low ruminal pH, enhancing the effects of EO. According to Cardozo et al. (2005) [
33], cinnamon EO and its main component, cinnamaldehyde, increase C
2:C
3 ratios in a rumen incubation medium with pH 7.0 while in a medium with pH 5.5, it causes a reduction in the C
2:C
3 ratio. Calsamiglia et al. (2007) [
37] also found that thymol EO was more effective at pH 5.5 than at 6.5.
Castillejos et al. (2006) [
24] in a 24 h in vitro assay using a substrate with 60% forage evaluated eugenol, guaiacol, limonene, thymol and vanillin EO at doses of 5, 50, 500 and 5000 mg/L. The effects observed by these authors depended on the doses used. The dose of 500 mg/L of thymol EO reduced the total concentration of SCFA (−28.5%), the proportion of propionate (−18.4%) and the concentration of NH
3-N (−31.9%) while there was an increase in the proportion of C
2 (+1.8%) and in the C
2:C
3 ratio (+35.5%).
In an experiment testing thyme, oregano and cinnamon EO, and their respective pure compounds—thymol, carvacrol and cinnamaldehyde—on rumen activity, Macheboeuf et al. (2008) [
38] observed results similar to those found in our experiment; however, cinnamon EO and its main compound cinnamaldehyde in concentrations lower than 3 mmol/L did not affect the production of CH
4 at 5 mmol/L. There was no production of CH
4 due to hydrogen accumulation in the headspace of the vial. Oregano, thyme, thymol and carvacrol EO were more toxic to ruminal microbiota, due to the lower dose needed (1, 1.5, 2.0 and 2.5 mmol/L, respectively) to inhibit the ruminal production of CH
4. This result reinforces the theory previously discussed that EO effects on ruminal microbiota depend on the dose used.
The variable CH4, expressed in mL/g IVDMD, represents the relationship between the production of net CH4 and IVDMD. The greater the value attributed to this variable, the greater the participation of net CH4 produced per gram of degraded dry matter (IVDMD) during the in vitro incubation process. Although the EO in our trial changed CH4 (mL/g IVDMD), there were no relationships observed between the production of CH4 and IVDMD. This can be attributed to the high standard error of the mean, since CH4, expressed as mL/g IVDMD, associates variability in gas measurements, CH4 liquid concentration, and residual non-degradable DM.