## 4 Discussion
### In Vitro Ruminal Fermentation  
The assessment of ruminal pH is a critical parameter for evaluating the stability and equilibrium of the rumen environment in ruminant animals, typically falling within the range of 5.0 to 7.5 [50]. In our investigation, we observed a dose-dependent effect of essential oils on ruminal pH, with the strongest impact observed at higher doses. Our findings align with the results reported by [PERSON] et al. [51], who noted no significant changes in rumen pH when low doses of clove bud oil were included into the cultures. However, at higher doses, a substantial increase in pH levels was observed. Similarly, [PERSON] et al. [52] conducted a study using a lower dose of clove essential oil (100 uL/L) and did not observe any noteworthy alterations in rumen pH levels. This lack of effect may be attributed to the relatively low concentration of clove essential oil used in their study. The observed increase in ruminal pH in our study can be attributed, at least in part, to the decrease in TVFA production and NH\({}_{3}\)-N concentrations. It is well-established that ruminal pH is influenced by the production and utilization of VFAs, with lower pH values associated with higher VFA concentrations [53]. The advantageous dose-dependent effects of essential oils on in vitro concentrations of NH\({}_{3}\)-N observed in our study hold promise for improving protein metabolism and reducing nitrogen waste in ruminal fermentation. Excessive ammonia production in the rumen has been linked to inefficient utilization of dietary protein and elevated nitrogen excretion [54]. Our findings demonstrate that essential oils, particularly at higher doses, have the potential to optimize protein metabolism and minimize nitrogen waste. The reduction in ammonia concentrations observed in our study is consistent with previous research highlighting the ammonia-lowering effects of essential oils in ruminal fermentation. For example, [PERSON] and [PERSON] [55] conducted a study investigating the impact of various essential oils on ruminal fermentation and found a significant decrease in ammonia production upon the addition of clove oil. This reduction in ammonia levels can be attributed to the bioactive compounds present in essential oils, which have the ability to inhibit the activity of ruminal microbial enzymes responsible for ammonia release. The observed decrease in TVFA concentration in the rumen is accompanied by a concomitant reduction in pH levels and NH\({}_{3}\)-N concentration, indicating a decrease in overall diet fermentation [56]. Since VFAs serve as the primary source of energy for ruminants, a reduction in ruminal VFA production could potentially have negative nutritional implications if this effect manifests in live animals. In our study, the inclusion of SAG, PIN, and CLO in the cultures resulted in a significant reduction of acetate, which is the primary VFA associated with methane production. This finding suggests that the addition of these essential oils has the potential to modulate rumen fermentation, leading to a decrease in methanogen activity [57]. Moreover, our study revealed that all the treatments employed exhibited an increase in propionate concentrations, consequently leading to a notable reduction in the A:P ratio. These findings further support the hypothesis put forward by [PERSON] et al. [58] that the application of essential oils enhances propionate production, thereby limiting the availability of H\({}_{2}\) for methane production. The observed dose-dependent reducing effects of essential oils on IVDMD and IVNDFD in our study are consistent with previous findings that have reported on the negative impact of essential oils on ruminal nutrient digestibility. For example, [PERSON] et al. [59] investigatedthe effects of clove essential oil on ruminal fermentation and nutrient digestibility and observed a decrease in dry matter digestibility at a dose level of 300 ppm. Similarly, in another study conducted by [PERSON] et al.