Evaluation of the Effects of Mitigation on Methane and Ammonia Production by Using Origanum Vulgare L. and Rosmarinus Officinalis L. Essential Oils on in Vitro Rumen Fermentation Systems

The effects of increasing concentrations of oregano (Origanum vulgare L.) and rosemary (Rosmarinus officinalis L.) essentials oil (EO) on ruminal gas emissions were tested in vitro using 50 mL serum bottles. Each bottle contained a 200 mg substrate (alfalfa hay and corn meal 1:1) and a 20 mL solution composed of a buffered medium and rumen fluid (1:2). The percentage of ruminal fermentation products was quantified by an infrared analyzer. The reduction of total gas production was 6% and 9% respectively when using the 1.5 and 2.0 g/L oregano EO measurements. The reduction of methane production was 55%, 72% and 71% respectively with regard to the 1.0, 1.5 and 2.0 g/L oregano EO doses, while rosemary EO (2.0 g/L) reduced the methane production by 9%. The production of ammonia was significantly reduced (59%–78%) by all treatments with the exception of 12857 rosemary EO at the lowest dose. Dry matter and neutral detergent fiber degradability was reduced by most of the treatments (respectively 4%–9% and 8%–24%). The total volatile fatty acids (VFA) concentration was markedly decreased by oregano EO and was not affected by rosemary EO. Both EOs mitigated rumen fermentations, but oregano EO gave rise to the highest reduction in methane and ammonia production. However, further research is needed to evaluate the use of these essential oils as dietary supplements by taking into account the negative effects on feed degradability.


Introduction
Agriculture produces about 10%-12% of the total global anthropogenic greenhouse gas (GHG) emissions: it contributes about 9% of the carbon dioxide (CO2), 50% of the anthropogenic methane (CH4) and 93% of the ammonia (NH3) emissions [1].Carbon dioxide gives the reference value of global warming potential (GWP) but is the most important GHG due to its high concentration in the atmosphere.The total CO2 emissions from livestock production are related to indirect activities such as the transport of raw materials, feed production, fertilizers, pesticides and fossil fuel consumption for energy use.Carbon dioxide emissions from metabolic and respiratory activities are not considered in the total GHG amount because they are balanced by CO2 captured by plants used for animal feeding.For this reason, animal respiratory activities are not considered to be a CO2 source by the Kyoto Protocol.
Methane is characterized by a GWP of 25 times greater than that of CO2 related to a period of 100 years [2].Its concentration in the atmosphere is less than that of CO2 but, due to its high GWP, methane is the second most responsible for global warming effects; however, methane may be used for supplying low environmental impact energy plants [3][4][5].Livestock production is the second source of global methane emissions.Most of them derive from ruminant enteric fermentations, which are necessary for carbohydrate digestion (cellulose, hemicellulose, pectin and starch).Methane emitted by animals depends on many factors such as breed, production level, forage quality, feed intake, genetics and feed conversion efficiency.Ruminant animals produce significant amounts of CH4 (33% of global CH4 emissions) and contribute significantly to global warming through the emission of 4% of total GHG [6].
Livestock production and the related manure management are responsible for about 64% of total ammonia emissions from anthropic activities [2].Ammonia is generated from the rapid degradation of urea in urine and feces; animal diet can influence its emission by modifying the metabolic efficiency of nitrogen.Ammonia emissions in the atmosphere are what is mainly responsible for acid rain.The effects of this phenomenon are the weakening of vegetation growth and the eutrophication of lakes and rivers.
Methane and NH3 production represent not only an environmental hazard but also a loss of dietary energy and nitrogen that could potentially be redirected to milk and meat production [7,8].Methane generation is achieved by archaea through the reduction of CO2 with hydrogen that is produced by various ruminal bacteria during feed digestion.In contrast, the NH3 production process mostly involves proteolytic bacteria and a group of bacteria called "hyper ammonia-producing bacteria" responsible of feed protein digestion and deamination of amino acids.The overproduction of ruminal NH3 causes large amounts of NH3 and urea to be excreted with urine; it contributes to ground water pollution and GHG by nitrous oxide emission [9,10].
The complexity of rumen microbial ecosystems makes the manipulation of rumen microbial fermentations an important challenge for ruminant nutritionists.Several dietary strategies were suggested to mitigate CH4 and NH3 generation from ruminants without negatively affecting animal performance.For example, plant extracts such as essential oils (EOs) were widely evaluated as feed additives to improve rumen microbial metabolism, protein degradation, fermentation efficiency and to reduce CH4 and NH3 production.
The effects of essential oils (EOs) are due to their antimicrobial activity against ruminal microorganisms such as methanogenic archaea and hyper-ammonia producing bacteria.However, EOs also show adverse effects on fiber digestion.Despite an extensive evaluation in the last years, some aspects of the use of EOs in ruminant nutrition are still unknown.For example, EO composition in active compounds can be highly variable and can be influenced by factors such as plant species, stage of growth, parts of the used plant, soil composition, temperature, moisture stress and extraction method.Moreover, most of the literature reports only the most common commercial EOs tested with a limited range of treatments and low experimental doses.
EOs from Lamiaceae family herbs such as oregano (Origanum vulgare L.) and rosemary (Rosmarinus officinalis L.) are well known for their antioxidant, antimicrobial, and medicinal properties but only a few studies investigated their properties in mitigating rumen GHG production.
The aim of this study was to evaluate the effects of increasing doses of oregano and rosemary EOs on CH4 and NH3 production and fermentation characteristics by an in vitro system.

Essential Oils
The oregano and rosemary EOs used in this study were purchased by Essential Srl (Montopoli Val d'Arno, Italy).EOs composition was analyzed by gas chromatography method.The analyses were performed by a Hewlett Packard HP 6890, combined with HP ChemStation Software, equipped with a flame ionization detector (FID) and a fused silica capillary column (HP-5MS; 30 m × 0.25 mm i.d., 0.25 μm film thickness).The oven temperature was programmed at 40 °C for 7 min, then ramped at 10 °C/min to 270 °C and held constant for 20 min.The injector and detector temperatures were respectively 250 °C and 270 °C.The samples were injected using the splitless mode; helium was used as carrier gas (1 mL/min).The samples were dissolved in hexane to give 0.125 μL/mL solutions; the injection volume was 1 μL.The percentage compositions of the oil components were obtained by FID electronic integration at 270 °C by dividing each component's area by the total area of all components.The percentage values were the mean of three sample injections.

In Vitro Fermentations
The ruminal inoculum for in vitro incubations was collected at a slaughterhouse from the rumen of three Chianina bulls fed with a ration composed of (dry-matter-basis percentage) corn silage (18.5%), mixed grass hay (13.4%), wheat straw (9.5%), wheat flour middlings (13.8%), barley meal (22.8%), soybean meal (14.6%) and corn meal (7.4%).Rumen contents from each cow were strained through four layers of cheesecloth into a thermos leaving no headspace.The obtained fresh rumen fluid samples were immediately transported to the laboratory and combined in equal volume.The in vitro buffered medium [11] was anaerobically prepared by a continuous CO2 flow as described in [12].The rumen fluid was added to the buffered medium in a 1:2 proportion.The in vitro fermentations were carried out in 50 mL serum bottles in triplicate for each treatment including control.The solution and the serum bottles were continuously flushed with CO2 and 20 mL of the medium was dispensed into each bottle containing 200 mg ground feed substrate.The feed substrate was composed of alfalfa hay and corn meal in a 1:1 ratio.Oregano and rosemary EOs were used at doses of 0, 0.5, 1.0, 1.5, 2.0 g/L (0 g/L is the control dose).The serum bottles were incubated at 39 °C for 24 h and subjected to continuous oscillation.

Sampling and Measurement
After a 24-hour incubation, gas pressure and gas composition (in terms of CO2, CH4, NH3) were recorded by an ABE_1500 (A.B.Energy Srl, Cossato, Italy) infrared portable gas analyzer, currently used by a renewable energy power plant [13][14][15][16][17].As well as the energy production plant monitoring, the infrared method for gas analyses was also used in previous lab scale experiments, which provided the evaluation of methane concentration due to rumen emissions [18,19].The analyzer uses an infrared (IR) detector and an electrochemical sensor.The first one is used to quantify CH4, CO2 and carbon monoxide (CO), which are characterized by high infrared absorption coefficients at low temperatures.The second one is used to quantify oxygen (O2) by electrochemical cells (Figure 1).NH3 and hydrogen sulfide (H2S) cannot be quantified by a standard configuration and an additional calibration of the electrochemical sensor is performed to trace these two compounds.The accuracy of the electrochemical and IR sensors is about 1.5%.All gas components are displayed together on a ¼ VGA LCD monitor (Table 1).The measurement range is 0%-100% volume for CH4, 0-20,000 ppm for CO, 0%-25% volume for O2.These characteristics and its lightness and practicality allowed use of the analyzer, typically suitable for on-site measurements, for gas detection in in vitro fermentation systems.The produced gas was vacuum sampled at the end of the fermentation phase by the instrument through a small plastic tube and a needle inserted into each bottle's rubber stopper.The content of each bottle was filtered through filter bags (Ankom Technology, Macedon, NY, USA; 50 µm pore size) to determine the degradability of residual feed substrate.The filtrates were sampled for the determination of volatile fatty acids (VFA) and stored at −80 °C before the analyses.VFA concentration, apparent dry matter (DM) degradability and neutral detergent fiber (NDF) degradability were determined as previously described by [20].

Statistical Analysis
Data on rumen fermentation characteristics were analyzed by SAS ANOVA procedure [21], which included EO type (oregano or rosemary) and dose level (0, 0.5, 1.0, 1.5 and 2.0 g/L) as fixed factors as well as their interaction.Differences between treatment means were determined by Tukey's test.Data were reported as least-squares means ± standard error.Differences were considered to be significant when p ≤ 0.05.

Effects on Gas Production and Feed Degradability
The effects of different doses of oregano and rosemary EOs on in vitro gas production and feed degradability are presented in Table 3.Total gas production decreased (p < 0.001) by about 6% and 9% respectively with the addition of 1.5 and 2.0 g/L oregano EO doses.Table 3. Effects of increasing doses of oregano and rosemary EOs on in vitro total gas (mL), methane (mL), ammonia (ppm), carbon dioxide (mL) production and degradability (%) of feed substrate.ANOVA revealed a significant (p < 0.001) interaction between treatment and dose: CH4 and CO2 productions were affected by 1.0, 1.5 and 2.0 g/L oregano EO doses (CH4 reduction, in particular, ranged from 55% to 72%), while rosemary EO reduced CH4 production only at the highest dose (−8.5%) and had no effect on CO2 concentration.Several studies documented the effect of EOs in reducing in vitro CH4 production through a direct inhibition of methanogenic archaea and/or an indirect depression of some microbial metabolic processes involved in methanogenesis [24].Macheboeuf et al. [25] showed that CH4 production was markedly inhibited for oregano EO doses of 0.45 and 0.75 g/L (respectively −63% and −97%).Roy et al. [26] obtained gas and CH4 production reductions at a 0.6 g/L oregano EO level.In contrast, no effect on gas production was found by rosemary EO but a reduction in CH4 production was observed for a 0.03 g/L level.EOs from garlic, cinnamon, eucalyptus, peppermint, juniper berry showed strong inhibitory activity on in vitro CH4 production comparable to the one obtained by oregano EO in the present study [25,[27][28][29][30].At present, few in vivo studies were conducted to evaluate the effects of these compounds on CH4 production.Some in vivo tests confirmed the potential of these plants and their extracts in CH4 mitigation.Hristov et al. [31] demonstrated a CH4 production reduction (−16.5, −11.7 and −13.6 g of methane per kg of DM intake) by three different doses of dietary oregano leaves (respectively 250, 500 and 750 g/animal per day) in dairy cows.Tekippe et al. [32] found similar results in dairy cows fed by oregano leaves at a 500 g/d rate (40% reduction in methane production).

Control
All treatments, except for rosemary EO at the lowest dose (0.5 g/L), reduced NH3 production (p < 0.01) in a dose-dependent manner (59%-78%), but DM and NDF degradability (p < 0.001) were reduced to the same extent (respectively 4%-9% and 8%-24%).The effects on DM and NDF degradability were more marked by the addition of oregano EO.Several works demonstrated that EOs can reduce NH3 concentration and protein deamination by inhibiting hyper-ammonia producing bacteria [33].Cardozo et al. [34] obtained a NH3 concentration decrease using 0.03 and 0.3 g/L oregano extract doses.Another in vitro study showed the capability to decrease NH3 production by 0.6 g/L oregano and rosemary EOs [26].In vitro NH3 concentration was also decreased by a combination of EOs composed of thyme, oregano, cinnamon and lemon applied at 0-0.5 g/L levels along with fumarate [35].Lin et al. [36] observed a NH3 inhibition with a mixture of EOs composed of clove, oregano, cinnamon and lemon in sheep (1 g/day).Contrasting results on in vivo NH3 production were found by Hristov et al. [31] and Tekippe et al. [32] by feeding dairy cows with a supplementation of oregano leaves.However, as confirmed by the present study, the positive effects of EOs on CH4 and NH3 production are frequently associated to negative effects on feed digestibility, especially at the highest doses [28,37,38].For example, Patra and Yu [30] observed a methane production reduction and a DM and NDF degradability decrease using a 1.0 g/L dose of EO from clove, eucalyptus, garlic, oregano and peppermint; the most pronounced effects were determined by oregano EO.The inhibition of feed digestion is due to the unselective antimicrobial activities of EOs that affect a wide range of microbial sub-populations such as, for instance, cellulolytic bacteria.In fact, Patra and Yu [30] found a decrease in the abundance of rumen archaea and protozoa but also in that of cellulolytic bacteria by all the tested EOs (clove, eucalyptus, garlic, oregano and peppermint).Most of EOs exhibit a wide range of antibacterial activity although Gram-positive bacteria are more sensitive than Gram-negative ones [39].Small molecular weight compounds, such as carvacrol and thymol (major constituent of oregano and thyme EOs), have the ability to destroy the Gram-negative bacteria's additional membranes by removing lipopolysaccharide membranes and increasing the permeability of cytoplasmic membranes [40].For this reason, these compounds show an antibacterial activity stronger than the other EO compounds [40].The abundance and activity of rumen fungi, protozoa and viruses can be also affected by EOs [33].A novel study is here presented which consists of the original comparison between the effects of oregano and rosemary EOs with known terpene composition on in vitro rumen fermentations and feed degradability.The presented results should confirm that high levels of carvacrol in oregano EO composition are responsible of its marked antimicrobial activity.

Effects on Production of Volatile Fatty Acids
The increasing doses of oregano and rosemary EOs showed different effects on total VFA concentration and molar percentage of individual fatty acids (see Table 4).A significant (p < 0.001) interaction between treatment and dose was observed: total VFA concentration was markedly reduced by 1.0, 1.5 and 2.0 g/L oregano EO doses; it was not affected by the tested rosemary EO doses.The molar proportion of acetic acid was increased (p < 0.001) only by the highest rosemary EO doses (1.5 and 2.0 g/L).No difference was shown between treatments and control on molar proportion of propionic and n-valeric acids.In contrast, the concentration of butyric acid was increased by the two lowest oregano EO levels and reduced by the two highest rosemary EO levels (p < 0.001).Isobutyric acid and acetic/propionic acid ratios were affected (p < 0.001) by rosemary EO only at a 2.0 g/L level.The lowest isovaleric acid molar proportions were (p < 0.001) obtained by 1.5 and 2.0 g/L oregano EO doses and 2.0 g/L rosemary EO dose.The 2.0 g/L oregano EO dose determined high concentrations of capronic (p < 0.01) and heptanoic (p < 0.05) acids.As described in the literature, supplementation with EOs can cause contradictory effects on total VFA concentration [41].Natural feed additives such as EOs can be considered useful in ruminant nutrition when they determine an increase of total VFA and propionic acid production and a decrease of the acetic/propionic acid ratio [40].Several studies demonstrated that these compounds have a detrimental effect on the total VFA production along with a reduction of feed digestion, especially with high EO doses.High oregano EO doses seem to have a strong inhibition activity on rumen fermentation and consequently negative effects on VFA concentrations [25].Busquet et al. [42] studied the effects of various plant extracts (anise, cade, capsicum, cinnamon, clove, bud, dill, fenugreek, garlic, ginger, oregano, tea tree and yucca) supplied at different doses on 24-hour in vitro ruminal fermentations.These authors observed that at the highest concentrations, most treatments decreased the total VFA production, as possible reflection of decreased feed digestion.Similar results were reported by Castillejos et al. [43].Instead, Patra and Yu [30] found that VFA concentration was affected only by oregano and clove EOs while the supplementation of EOs in sheep diets did not show any effect on VFA production [20,36].Some researchers showed that some EOs have positive effects on VFA molar proportions by decreasing the acetate production and increasing propionate production [44][45][46].In accordance with the obtained results, many studies showed that the acetate/propionate ratio was increased [25,38,47] or not affected [29,48] by EOs.The effects of EOs on VFA production may depend on the ruminal fluid pH.Cardozo et al. [34] found that the effects of some EOs on rumen VFA profiles were more pronounced at low rumen pH; they suggested that pH is able to affect the dissociated or undissociated status of EO molecules.

Conclusions
The results of the proposed investigation showed that oregano EO, at the highest used concentrations, is a potent inhibitor of ruminal CH4 and NH3 production; it is probably due to the antimicrobial properties of carvacrol, its major compound.In contrast, rosemary EO showed limited effects on CH4 mitigation but a strong inhibition activity on NH3 production.Unfortunately, the most effective EO concentrations on GHG mitigation have also demonstrated adverse effects on feed degradability and VFA production as result of their marked and non-specific antimicrobial activity.Further research is needed to evaluate the use of oregano and rosemary EOs as dietary supplements by taking into account their unfavorable effects on feed degradability.Their biological activity depends on their composition and their composition is highly variable; thus, only a deep knowledge of EO nature, structure and purity can help to understand their favorable and/or unfavorable properties.
fatty acids; SEM: standard error of the mean; T: treatment; D: dose; TxD: treatment x dose; a-d Means with different letters within a same row differ significantly.

Table 1 .
Range and accuracy of measurements performed by ABE_1500 gas analyzer.

Table 2 .
Oregano and rosemary EO composition.Components are listed in order of their elution from a HP-5MS column; c Identification: by comparison of the mass spectrum with those of the NIST98 library computer (99% matching); d RI: Retention index, by comparison with those reported from NIST databank.Linear retention indices were determined relative to the retention times on HP-5MS columns of homologous series of C 5 -C 20 alkanes by a Van den Dool and Krantz equation; ND: not detected.
a Percentage obtained by FID peak-area normalization.Values are presented as the mean ± SD (n = 3); b

Table 4 .
Effects of increasing doses of oregano and rosemary EOs on in vitro total VFA concentrations (mM) and molar percentage (%) of each fatty acid.