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Part Two – Effects of Oregano Essential Oil On in Vitro Ruminal Fermentation, Methane Production, & Ruminal Microbial Community

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Ruminant livestock production has been identified as a significant contributor to greenhouse gas emissions (Gerber et al., 2013). Seventy-one percent of the methane produced by enteric fermentation originates from ruminal fermentation (Gerber et al., 2013). In recent years, numerous strategies to mitigate methane emissions through the modulation of ruminal fermentation have been reported. Feeding natural feed additives has shown promising results (Hristov et al., 2013; Knapp et al., 2014). Among these natural additives, most are sourced from several plant types as secondary metabolites that have been reported to be useful in animal feeding (Wallace, 2004; Kamra et al., 2006), such as saponins, tannins, and essential oils (EO), which have demonstrated antimicrobial activities (Bakkali et al., 2008). Among these secondary metabolites, EO gets the most attention and are being investigated as alternatives to antibiotics in animal nutrition (Greathead, 2003; Froehlich et al., 2017).

In addition, EO and their bioactive compounds have been confirmed to modify ruminal fermentation by enhancing the efficiency of energy utilization while decreasing methane emissions (Tekippe et al., 2010; Joch et al., 2016). Essential oils have also been investigated as potential modifiers of ruminal biohydrogenation of dietary lipids to enhance the healthful characteristics of milk and meat (Durmic et al., 2008; Lourenço et al., 2008). Oregano (Origanum vulgare L.) is an herb with higher antioxidant capacity than other medicinal herbs (Dragland et al., 2003; Matsuura et al., 2003). The primary constituents of oregano EO (OEO) are carvacrol, γ-terpinene, thymol, p-cimene, and linalool, depending on origin and type of OEO (Sivropoulou et al.,1996; Baser, 2002). Oregano EO has been reported to have the second-highest oxygen radical absorbance capacity compared with the clove (the highest), followed by cinnamon, ginger, and rosemary EO (Bentayeb et al., 2009).

Thymol is a monoterpene with potent antimicrobial activity against a wide range of gram-positive and gram-negative bacteria and is one of the most actively researched EO (Burt, 2004). Carvacrol is a phenolic compound similar to thymol found in oregano, which also has potent antimicrobial activity. Busquet et al. (2005b) reported that carvacrol (2.2 mg/L) decreased large-peptide concentrations and increased ammonia nitrogen concentrations two h after feeding, using an in vitro long-term ruminal continuous culture system. Wang et al. (2009) evaluated a commercial OEO preparation (active chemical compounds: carvacrol, thymol) that demonstrated approximately a 12% decrease in methane production in sheep, similar in magnitude to methane production levels with flavomycin and Yucca schidigera (i.e., saponins). Oregano leaf material as an anti-methanogenic plant product, with no adverse effects on ruminal fermentation or NDF degradability, has been reported in vitro (Tekippe et al., 2010).

Oregano EO was tested using a single dose and reduced ruminal methane production by positively improving the total-tract apparent digestibility of dietary nutrients (Liu et al., 2017). The study objectives were to verify our previous results and to evaluate the inclusion rates of OEO to aid in determining the optimal feeding rate. Therefore, the specific study objective was to assess the effects of increasing OEO supplementation rates on ruminal in vitro fermentation, methane production, and ruminal bacterial communities. The hypothesis was that OEO would reduce fermenter methane production while improving feed digestibility and fermentation characteristics.


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Effects on Rumen Fermentation Characteristics

The digestibility of DM, NDF, and ADF increased quadratically (P < 0.03) with increasing OEO inclusion rates. In slight contrast, DM and NDF digestibility were highest for OEO2, whereas ADF digestibility was highest for OEO3 compared with CON, with the remaining treatments being intermediate and similar (P > 0.05). It thus appears that OEO3 would be the optimal feeding rate for future in vivo studies. Fermenter pH demonstrated a linear (P < 0.05) increase with increasing OEO inclusion rate, with the highest pH observed for the highest inclusion rate of OEO (OEO4). In contrast with fermenter pH, NH3-N concentrations demonstrated a quartic response (P < 0.01) with increasing OEO inclusion rates, such that OEO2 had the lowest (P < 0.05) NH3-N concentrations compared with the CON and OEO1 fermenters. The inclusion rate of OEO demonstrated mixed effects on fermenter VFA concentrations. Microbial CP concentrations demonstrated a quadratic response (P < 0.10), such that the OEO2 fermenter had the highest MCP concentration compared with fermenters CON, OEO3, and OEO4.

The MCP concentrations were inverse of the fermenter ammonia concentrations, indicating that ammonia was being incorporated into the synthesis of MCP. Total concentrations of VFA, propionate, butyrate, isovalerate, and acetate-to-propionate ratio demonstrated trends (P < 0.10) or significant (P < 0.05) quartic responses to increasing OEO inclusion rate, decreasing to lower concentrations for OEO4 compared with CON fermenters. Acetate demonstrated a quadratic response (P < 0.10), with OEO4 decreasing to a lower concentration compared with fermenters CON or OEO1. Isobutyrate concentrations linearly decreased (P < 0.01) with increasing OEO inclusion rate. As the first OEO inclusion rate was added to the fermenters, most VFA remained similar (P > 0.10) to CON, but further increases in OEO inclusion rate decreased VFA concentrations (P < 0.05), meaning that very high OEO inclusion rates can become detrimental or inhibitory to VFA production.

Effects on Total Gas and Methane Production

Total 24-h gas production demonstrated a quartic (P < 0.10) response, decreasing to lowest for OEO2 then increasing with increasing OEO inclusion rate, whereas 24-h total methane production and methane/total gas were linearly decreased (P < 0.05) with increasing OEO inclusion rate, such that OEO3 had the lowest methane production and methane/total gas compared with the CON and OEO1 fermenters. However, as total gas and methane decreased, methane as a percentage of total gas production declined faster as the OEO inclusion rate increased. Methane dropping at a greater rate than total gas production should direct more energy into VFA production.

Effects on Rumen Bacterial Community

A total of 379,768 raw bacterial 16S rRNA gene sequences were obtained. After quality control and filtering, 25,317 valid sequences were analyzed for each sample. A total of 4,410 OTU based on 97% sequence similarity were generated. The α diversity of 16S rRNA gene OTU was not altered by increasing OEO inclusion rate (P > 0.10;), except for Chao1, which demonstrated a linear (P < 0.05) reduction such that OEO3 fermenters were lowest (P < 0.05) compared with remaining fermenters. Based on the obtained OTU, a total of 24 genera were observed to have a relative abundance greater than 1% (Figure 1). The most abundant microbial genus detected via 16S rRNA gene sequence data was Prevotella, which demonstrated a linear (P < 0.05) response, with increasing OEO inclusion rate being most significant (P < 0.05) for OEO4 fermenters compared with CON fermenters and the remaining treatments being intermediate.

Several microbial genera were similar (P > 0.05) among all OEO inclusion rates. Firmicutes unclassified, Eubacterium, Lachnospiraceae unclassified, Bifidobacterium, and Oxobacter demonstrated quartic (P < 0.05) responses with increasing OEO inclusion rates that were not consistent across microbial genera. Firmicutes unclassified, Eubacterium, Bifidobacterium, and Oxobacter were lower for OEO1 fermenters compared with CON fermenters. In contrast, Lachnospiraceae unclassified was lower for CON, OEO2, and OEO3 fermenters compared with OEO1 fermenters, with the remaining treatments being intermediate. The microbial genera with more than 2% relative abundance demonstrate a greater relative abundance of Prevotella and Dialister while reducing (P < 0.010) Veillonellaceae unclassified, Porphyromonadaceae unclassified, and Firmicutes unclassified via linear, quadratic, cubic, or quartic responses with increasing OEO inclusion rate compared with CON.


Effects on Rumen Fermentation Characteristics

For a ruminant animal, most of the DM and fiber of a feed or ration is ruminally digested. Therefore, ruminal DM and fiber digestibility are appropriate indices for evaluating OEO effects (Lin et al., 2009). In the literature, the EO effects on nutrient digestibility by the rumen microbiome have been mixed. Some researchers have suggested that EO has no effects on fiber degradation but can reduce the degradation of readily degradable substrates, such as protein and starch, owing to OEO inhibition of amylolytic and proteolytic bacteria (Wallace et al., 2002; Hart et al., 2008). On the contrary, other authors have reported positive effects (Benchaar et al., 2006; Yang et al., 2007) or no effects (Meyer et al., 2009; Sallam et al., 2009) of EO on ruminal digestibility. In the current study, supplementation of 52 mg/L and 91 mg/L OEO increased in vitro fermenter DM, ADF, and NDF digestibility compared with CON, with digestibility increasing with increasing OEO inclusion rate to a point of being maximal at 52 or 91 mg/L.

These results corresponded to a linear decrease in VFA production, to the extent that a high OEO inclusion rate can be detrimental by reducing VFA concentrations. Why digestibility could be increased with a reduction in VFA may be related to the production of microbial extracellular enzymes enhancing DM and fiber digestibility (Priest, 1977), but the hydrolysis products were not fermented to VFA. These findings are in contrast with the results reported by Tager and Krause (2011). They reported that high levels of EO negatively affected ruminal fiber digestibility but did agree that they theoretically could reduce ruminal VFA production. The potential uses of EO as a ruminal fermentation modifier have been reported in the literature (Molero et al., 2004; Simitzis, 2017). Numerous in vitro experiments have reported that different types and doses of EO could inhibit NH3 concentrations (Cardozo et al., 2005; Lin et al., 2012; Lin et al., 2013b).

Hristov et al. (2013) also observed decreased NH3 and butyrate concentration when feeding dairy cows Origanum vulgare L. leaves (250, 500, and 750 g/cow). The various EO available as natural feed additives could be considered useful in ruminant nutrition by reducing ruminal ammonia N concentrations and protein deamination by inhibiting hyper-ammonia-producing (HAP) bacteria (Patra, 2011). The quartic response in decreasing ruminal ammonia nitrogen concentrations with increasing OEO inclusion rate observed in the present study likely resulted from the inhibition of proteolysis, peptidolysis, amino acid deamination, or an increase in microbial protein synthesis, or some combination of these factors—a premise substantiated by the reduced concentrations of isobutyrate and isovalerate reported by Patra and Yu (2012). In the present study, the pH increased linearly with increasing OEO inclusion rate, likely due to the reduction in VFA production, which agrees with previous studies (Lin et al., 2012; Patra and Yu, 2015a).

Several studies have observed that EO supplementation can reduce VFA concentrations (Busquet et al., 2006; Calsamiglia et al., 2007; Benchaar et al., 2008). Patra and Yu (2012) reported, using an in vitro test, that total VFA concentration was decreased by adding oregano and clove EO (0.25, 0.50, and 1.0 g/L). Additionally, a mixed EO blend (50 or 200 mg/L eugenol, carvacrol, citral, and cinnamaldehyde oils) fed to Hu sheep effectively reduced the acetate: propionate ratio, NH3-N, and total VFA concentrations (Lin et al., 2013a). As confirmed by the present study, total VFA, acetate, and butyrate concentrations were markedly reduced with increasing OEO inclusion rates. The propionate concentration and acetate: propionate ratio was increased with the addition of 52 mg/L of OEO. Even though VFA concentrations were reduced with increasing OEO inclusion rate, the propionate concentration increased and then decreased in a quadratic response. The inclusion of OEO caused a shift in ruminal fermentation for lower acetate and greater propionate concentrations.

Thus, it is plausible that OEO supplementation causes an ionophore-like shift in the ruminal microbial ecology. Several studies have demonstrated that some EO has positive effects on VFA concentrations by decreasing acetate production and increasing propionate production (Mohammed et al., 2004; Busquet et al., 2005a; Poudel et al., 2019). Poudel et al. (2019) demonstrated that feeding the same OEO product used in this study resulted in an increase in ruminal propionate concentrations from Holstein calves, along with an increase in Prevotellaceae. In the present study, supplementation with increasing OEO inclusion rates decreased isobutyrate and isovalerate concentrations. These results agree with the findings reported by Pinski et al. (2016), who suggest that different doses of OEO have different potencies to inhibit proteolysis and amelogenesis. Additional studies using HAP bacterial populations need to be conducted to elucidate the effects of OEO on amelogenesis.

Effects on Total Gas and Methane Production

In the present study, total gas production was linearly decreased by increasing OEO inclusion rate. These results agree with the in vitro results reported by Patra and Yu (2012) and Cobellis et al. (2016b) that supplementation with cinnamon bark oil, Ceylon cinnamon bark oil, and oregano oil demonstrated a pronounced inhibition of total gas production. The novel finding in our study is an increasing reduction in methane as a percentage of total gas production with a rising OEO inclusion rate. However, Agarwal et al. (2009) found an increase in gas production with the addition of 0.33 and 1.0 μL/mL of peppermint oil. These results demonstrate that the OEO type and dose may elicit different results. Numerous studies have reported potent inhibition in methane production by EO (Wang et al., 2009; Knapp et al., 2014; Cobellis et al., 2016b). For example, Macheboeuf et al. (2008) observed a 98% reduction in methane production using five mM of OEO or cinnamon EO.

Evaluating five different EO (clove, eucalyptus, garlic, oregano, and peppermint), Patra and Yu (2012) reported that methane production linearly decreased with increasing EO doses, with the most significant methane reduction (87%) using OEO at a dose of 1.0 g/L. Garlic oil can inhibit in vitro methane production (38.5%) with an amount of 167 μL/L (Pawer et al., 2014). The finding of Pawer et al. (2014) is an increasing methane reduction as a % of total gas production with a rising OEO inclusion rate. In the present study, methane production decreased linearly with increasing OEO supplementation, suggesting that OEO inhibits methane synthesis. The decrease in methane production with the addition of OEO is similar to the findings of Tekippe et al. (2011). Feeding lactating cows (8 cows) Origanum vulgare L. leaves (500 g/cow per d) led to an approximately 31% decrease in ruminal methane production.

The inhibition of methane production by OEO may be due to indirect or direct inhibition (or both) of methanogens via a decline in H2 production due to reduced acetate and butyrate and more propionate production (Cieslak et al., 2013; Kumar et al., 2014). Shifting ruminal fermentation to more propionate would inhibit hydrogen-producing bacteria, such as Ruminococcus albus, Ruminococcus flavefaciens, and protozoa (Cobellis et al., 2016b). Further studies are needed to elucidate the exact mechanism by which OEO reduces methane production.

Rumen Microbial Community

In previous studies, very few bacteria genera were analyzed (Fiorentini et al., 2013; Martínez-Fernández et al., 2014; Zhou et al., 2017), which prevented a deeper understanding of the influence of feed additives on ruminal bacterial communities. In the present study, 16S rRNA sequencing was used to examine OEO influence on the ruminal bacterial community. Ruminant animals have a very diverse bacterial community, especially at the species level, containing about 5,200 OTU (Kim et al., 2011). A similar result (4,410 OTU) was observed in our study. The phyla Firmicutes, Bacteroidetes, and Proteobacteria, were the prevalent bacteria in the current study, which agreed with previous studies (Zhou et al., 2017; Stewart et al., 2018; Yan et al., 2018). Antibacterial potency of OEO (containing phenol) may contribute to the antimicrobial activities of OEO (Ultee et al., 2002). Generally, gram-positive bacteria are thought to be more susceptible to EO than are gram-negative bacteria, due to the lack of a protective outer membrane surrounding the cell wall (Patra and Yu, 2012).

In the present study, although Firmicutes was not affected by OEO. Members of Firmicutes, Selenomonas, and Mitsuokella, gram-positive genera, were linearly decreased by OEO inclusion rates. Dialister and Lactobacillus were linearly increased by OEO inclusion rates, suggesting that these bacteria may play a key role in ruminal biohydrogenation (Huws et al., 2011; Patra and Yu., 2015b). In the present study, supplementation with 13 mg/L of OEO increased the relative abundance of Lachnospiraceae unclassified. The relative abundances of Lactobacillus were increased by the three highest OEO inclusion rates, suggesting that these bacteria may play a key role in ruminal biohydrogenation (Huws et al., 2011; Patra and Yu., 2015b). Therefore, changes in microbial compositions via OEO inclusion may be associated with changes in the ruminal biohydrogenation process (Ramos-Morales et al., 2013).

Thoetkiattikul et al. (2013) reported that Bacteroidetes was a major non-cellulosic plant constituent degrader in the rumen. Most Bacteroidetes bacterial strains are hemicellulolytic, proteolytic, or amylolytic (Evans et al., 2011). The relative abundance of the top 2 most abundant bacteria of the Bacteroidetes family (Prevotella and Dialister) were increased by OEO, possibly due to reduced competition from other bacteria that are inhibited by OEO (Patra and Yu, 2015b). Prevotella, a gram-negative genus of Bacteroidetes, linearly increased with OEO inclusion rates, maybe due to reduced competition from other bacteria, which are inhibited by OEO (Patra and Yu, 2015b). In addition, we observed that Prevotella was the dominant genus among all treatments, in accordance with some previous in vivo studies (Jami et al., 2013; Thoetkiattikul et al., 2013; Paz et al., 2016). It has been suggested that EO could reduce ruminal protein degradation and ammonia concentrations (McIntosh et al., 2003; Patra, 2011).

We also report that OEO supplementation significantly lowered ammonia nitrogen, which agrees with previous studies that reported that EO decreased ruminal ammonia concentrations (Patra and Yu, 2012; Patra and Yu, 2014). The methanogens Euryarchaeota are a phylum belonging to the archaea community; in the current study, Euryarchaeota showed very low relative abundance and were decreased at all levels of OEO inclusion, consistent with the decrease of CH4 production in OEO treatments. These results indicate that ruminal methane production may be much more influenced by the relative abundance of archaea rather than by the microbial population structure (Wallace, 2004; Duarte et al., 2017). We noted that Fibrobacteres accounted for less than 1.0% of the sequences among all treatments.

This result is similar to a study by Patra and Yu (2015b), reporting 1 OTU Fibrobacteres with the addition of 0.5 g/L OEO. In addition, the cellulolytic Fibrobacteres bacteria decreased for the 52 and 91 mg/L OEO treatments, which may be positively correlated with acetate concentration (Varzaneh et al., 2018). In the present study, OEO supplementation also reduced a few other bacteria, such as Verrucomicrobia, Synergistetes, Spirochaetes, and Planctomycetes. However, the rumen metabolism of these genera are not well understood, and further studies are needed to elicit their functions.


Previous studies using 16S rRNA analysis demonstrate that OEO supplementation can influence ruminal bacterial communities to increase the digestibility of dry matter and fiber while shifting fermentation to reduce molar concentrations of acetate and butyrate to increasing propionate. The modulation of fermentation by OEO inclusion rate altered many ruminal bacterial genera associated with feed digestibility and ruminal fermentation characteristics. However, many ruminal bacteria remain to be cultured, cautioning that the results obtained in the present study might have limitations. Future in vivo studies is needed to optimize OEO inclusion rate so that effective methane mitigation can be achieved without altering ruminal feed digestion and fermentation. However, an optimal OEO feeding rate for future animal studies appears to be 52 mg/L for mature ruminants.

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