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Part One – Effects of oregano essential oil on in vitro ruminal fermentation, methane production, and ruminal microbial community

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Different inclusion rates of oregano essential oil (OEO) were investigated for their effects on ruminal in vitro fermentation parameters, total gas, methane production, and bacterial communities. Treatments were (1) control, 0 mg/L of OEO (CON); 13 mg/L (OEO1); 52 mg/L (OEO2); 91 mg/L (OEO3); and 130 mg/L (OEO4), each incubated with 150 mL of buffered rumen fluid and 1,200 mg of substrate for 24 h using the Ankom in vitro gas production system (Ankom Technology Corp., Fairport, NY). Treatment responses were statistically analyzed using polynomial contrasts. Digestibility of DM, NDF, and ADF increased quadratically with increasing OEO inclusion rates. Digestibility of DM and NDF were highest for OEO2, whereas ADF digestibility was highest for OEO3, compared with CON, with the remaining treatments being intermediate and similar. Ammonia nitrogen concentrations decreased from CON at a quadratic rate with increasing OEO inclusion rates, and OEO2 had the lowest concentration compared with the other groups.

Total VFA, acetate, propionate, butyrate, valerate, and isovalerate concentrations linearly decreased with increasing OEO inclusion rates. Total gas production levels by CON and OEO4 were greater than those of OEO1, OEO2, and OEO3 in a quadratic response, and methane production linearly decreased from CON, compared with OEO4, at a decreasing rate with OEO inclusion rates. As determined by 16S rRNA sequencing, the α biodiversity of ruminal bacteria was similar among OEO inclusion rates. Increasing OEO inclusion rates linearly increased the relative abundance of Prevotella and Dialister bacteria. Several bacteria demonstrated different polynomial responses, whereas several bacteria were similar among increasing OEO inclusion rates. These results suggested that OEO supplementation can modify ruminal fermentation to alter VFA concentrations and reduce methane emissions by extensively altering the ruminal bacterial community, suggesting an optimal feeding rate for future animal studies of approximately 52 mg/L for mature ruminants.


This limited series of the occasional e-letters are comprised of (2) two articles. They will appear fortnightly and are published during March and April, though they will be accessible through our social media pages.


Feeding and Management

All animal-handing protocols were approved by the Gansu Agricultural University Animal Care and Use Committee Guidelines (approved ID: 2012-2-159), following the Chinese Standards for the Use and Care of Research Animals (He et al., 2016). Three German Merino sheep × local sheep crossbreed rams (initial live BW = 56.68 ± 2.14 kg) fitted with permanent rumen cannulas were used as ruminal inoculum donors. Sheep were selected as the inoculum source due to being readily available, and Yáñez-Ruiz et al. (2016) indicated no differences between cattle and sheep when conducting in vitro experiments. Cows were offered a 65.5:34.5 forage-to-concentrate ratio (DM basis, %) based on corn silage, ground corn, protein sources (soybean, rapeseed, and cottonseed meals), minerals, and vitamins. The nutrient composition of the diet (%, DM basis) was approximately 15.1% CP and 51.2% NDF, to meet or exceed the nutrient requirements guidelines for sheep (NRC, 2007). Sheep were fed twice daily at 0800 and 1800 h, before and throughout the duration of the experiment, and were given ad libitum access to water.

Oregano Essential Oil Treatments

The OEO was supplied as a dry granular powder (Rum-A-Fresh, Ralco Inc., Marshall, MN) contain ing approximately 1.3% OEO, along with lactic acid, cobalt carbonate, and clinoptilolite as a carrier. Five inclusion rates of OEO were evaluated: 0 (CON), 13 mg/L (OEO1), 52 mg/L (OEO2), 91 mg/L (OEO3), and 130 mg/L (OEO4), adjusted for the amount of OEO in the dry granular product added to the in vitro fermentation medium.

In Vitro Fermentations

Whole ruminal contents were obtained approximately two h after the morning feeding from 3 sheep in equal proportions, transported to the laboratory in a prewarmed and sealed flask, and immediately mixed. The ruminal fluid for incubations was obtained after filtering through 4 layers of 100 mm × 100 mm medical gauze (Winner Inc. Ltd., Shenzhen, China) under constant CO2 flushing. The in vitro experiment was carried out using the Ankom RFS gas production system (Ankom Technology Corp., Fairport, NY). The system comprised 24 fermenters having a 250-mL volume, containing 1,200 mg of feed substrate (same ration fed to donor sheep, ground to pass through a 1-mm sieve) and the corresponding OEO inclusion rate. Each treatment had four bottles and four blank bottles (rumen fluid only) with no feed substrate, to correct for background gas production. Each fermenter was filled with 150 mL of ruminal inoculum, prepared as a 1:2 ratio of rumen fluid and artificial saliva. The artificial saliva was prepared anaerobically as described by Menke and Steingass (1988). All fermenters were incubated at 39°C for 24 h in a mild shaking water bath (SPH-110X24, Shiping Ltd., Shanghai, China).


Headspace gas (5 mL) was collected from each fermenter bottle at 24 h, using a sealed gas injection needle, for determination of methane concentrations. At the end of the 24-h incubation, fermenters were opened, and pH was immediately measured using a pH meter and glass electrode (Type CG 842, Blueline 14 pH, Schott Instruments, Weilheim, Germany). A liquid sub-sample was collected from each fermenter and stored in microcentrifuge tubes for later determination of VFA, ammonia nitrogen (NH3-N), microbial crude protein (MCP), and bacterial community determinations. Samples were stored at −20°C for VFA and ammonia nitrogen and at −80°C for MCP and bacterial community analyses, respectively, until analyzed. The residues of each fermenter were collected after centrifugation at 4,000 × g for 10 min at room temperature to determine the DM, NDF, and ADF digestibility (TGL16, Cence Ltd., Changsha, China).

Chemical Analysis

The VFA concentrations were analyzed using a gas chromatograph (GC-2010, Shimadzu, Kyoto, Japan) following the procedures described by Hu et al. (2005). The NH3-N concentration was analyzed spectrophotometrically (UV-120-01, Shimadzu) using the procedures described by Chaney and Marbach (1962). The MCP was determined following the procedures of Lin et al. (2013a). Feed substrate and residues DM were determined following AOAC International (2016) procedures. The NDF and ADF in the feed substrate and residues were determined according to the methods described by Van Soest et al. (1991). Methane production was analyzed via gas chromatography (GC-2010, Shimadzu) equipped with a flame ionization detector (FID-2010, Shimadzu; Hu et al., 2005). The column (HP-INNOWAX, 19091N-133, Agilent Technologies, Santa Clara, CA) of the GC was 30 m × 0.25 mm × 0.25 μm in size, and the temperature was 80°C.

The gas flow rates for nitrogen, hydrogen, and air were 30, 25, and 400 mL/min, respectively. The front injects port, column oven, and detector temperatures were 50, 375, and 200°C, respectively. The gas pressure was automatically recorded during incubation (Ankom Technology Corp.). The total gas production at 24 h was calculated based on the ideal gas equation: Vx = PkPa × 1.3815886 (Ankom operations manual), where Vx (mL) = gas volume at 39°C, and PkPa = gas pressure.

DNA Extraction, PCR Amplification, and 16S rRNA Sequencing

Total genomic DNA from ruminal samples stored at −80°C was extracted using the EZNA Stool DNA Kit (D4015, Omega Bio-tek Inc., Norcross, GA), following the manufacturer’s instructions. The V3 to V4 region of the prokaryotic (bacterial and archaeal) small-subunit (16S) rRNA genes were amplified using slightly modified universal primer versions 338F (5’-ACTCCTACGGGAGGCAGCAG-3’)  and  806R (5’-GGACTACHVGGGTWTCTAAT-3’; Fadrosh et al., 2014), where the barcode is an 8-base sequence unique to each sample on the 5 primer ends and sequencing universal primers. Amplification via PCR was performed in a total volume of 25 μL of reaction mixture containing 25 ng of template DNA, 12.5 μL of PCR Premix, 2.5 μL of each primer, and PCR-grade water to adjust the volume. The PCR conditions to amplify the prokaryotic 16S fragments consisted of an initial denaturation at 98°C for 30 s; 35 cycles of denaturation at 98°C for 10 s, annealing at 54°C for 30 s, and extension at 72°C for 45 s; and then final extension at 72°C for 10 min.

The PCR products were confirmed using 2% agarose gel electrophoresis. The PCR products were purified using AMPure XT beads (Beckman Coulter Genomics, Danvers, MA) and quantified by Qubit (Invitrogen, Carlsbad, CA). Amplicon sequencing was conducted on an Illumina MiSeq 2 × 3 platforms to generate overlapping paired-end 2 × 300 bp (Bionew Ltd., Hohhot, China).

Bioinformatics and Analyses

All reads were processed and analyzed using the following procedures. First, paired-end reads were assigned to samples based on their unique barcode and truncated by cutting off the barcode and primer sequence. Paired-end reads were then merged using FLASH 1.2.7 (Magoč and Salzberg, 2011). Quality filtering of the raw tags was performed under specific filtering conditions to obtain high-quality clean tags according to the fqtrim (version 0.94, National Center for Biotechnology Information, Bethesda, MD). Chimeric sequences were filtered using USEARCH software based on the UCHIME algorithm (Edgar et al., 2011). Bacterial operational taxonomic units (OTU) were selected using USEARCH (version 2.3.4; Edgar, 2010), applying 97% sequence similarity thresholds. Representative sequences for each OTU were chosen using Quantitative Insights Into Microbial Ecology (QIIME; Wemheuer et al., 2014) programs (version 1.9.1), which were assigned from the Ribosomal Database Project for taxonomic classification using the BLAST approach (Mitra et al., 2011).

Alpha diversity analysis, including abundancebased coverage estimators (ACE), Good’s coverage, Chao1, Shannon, Simpson, and observed OTU indices, were calculated with QIIME 1.9.0 according to Wemheuer et al. (2014).

Statistical Analyses

All data were checked for normality and outliers using the UNIVARIATE procedure of SAS (version 9.4, SAS Institute Inc., Cary, NC) before any statistical analyses were conducted. We performed ANOVA using the R package (version 3.4.1, R Core Team, 2017) or SAS. The Mann-Whitney U test was applied to determine the significance of α diversity (Zeng et al., 2015). The data of ruminal fermentation parameters and nutrient digestibility were statistically analyzed using the PROC MIXED procedure of SAS. The statistical model used was as follows: Yij = μ + Ri + Tj + eij, where Yij = dependent variable, μ = overall mean, Ri = replication, Tj = treatment, and eij = random error. Treatment effects were considered fixed, and replication was considered to be random effect. Polynomial contrasts were used

to test the linear, quadratic, cubic, and quartic effects of treatments (i.e., OEO inclusion rate). Differences among treatments were separated using the PDIFF statement of SAS. Statistical significance was set to P< 0.05, and a tendency of difference was declared at P< 0.10.


Capper et al. (2011) reported that consumers often have the perception that modern beef production has an environmental impact far greater than that of historical systems. Consumer perception is that improved efficiency is being achieved at the expense of increased greenhouse gas emissions. They concluded that beef production in 2007 required considerably fewer resources than the equivalent system in 1977, with 69.9% of the animals, 81.4% of the feedstuffs, 87.9% of the water, and only 67.0% of the land required to produce 1 billion kg of beef. Waste outputs were similarly reduced, with the 2007 beef production systems producing 81.9% of the manure, 82.3% of the methane, and 88.0% of the nitrogen oxide per billion kg of beef compared with equivalent systems in 1977. The carbon footprint per billion kilograms of beef produced in 2007 was reduced by 16.3% compared with equivalent systems in 1977 (Capper et al., 2011). Hristov et al. (2013a, b) reviewed the literature as to methods to reduce methane and nitric oxide emissions from cattle. They reported the work of Sauvant and Giger-Reverdin (2009) who showed that as DM intake and the concentrate portion of the diet increase, enteric CH4/DM intake declines.

Hristov et al. (2013a, b) and Hyland et al. (2017) concluded that alterations in the production system that result in improved utilization of the animal’s diet and improved system efficiency especially through improved fertility will reduce enteric CH4 and N2O emissions.  We will see in the next E-letter the effect of oregano oil on different key points influencing the production of these gaz. Stay tuned! We want to proceed further, more in-depth on this controversial subject. Therefore, please follow us on social media and join us on the (15th) fifteenth of the month for Part (2!) two to learn more about the environmental impact of beef production.  Thereafter, please join us on the (1st) first and (15th) fifteenth of each month for our fortnightly delivery of insightful, informative must-reads from some of the world’s scientific thinkers. Selected by our editors is a collection of current topics with a profound ability for beneficial improvements, guidelines, and process practices. Thank you for reading our publication entirely; please share it with others who also care. We look forward to your comments and having you with us again fortnightly; we will be thrilled in having you with us; thus, we will take your trust in us with great honor and appreciation.