Lipid supplementation is a promising strategy for methane mitigation in cattle and has been evaluated using several different lipid sources. However, limited studies have assessed the effect of temperature on methane emissions from cattle and changes in incubation temperature have also not been extensively evaluated. The aim of this study was to evaluate the combined effect of pequi oil (high in unsaturated fatty acids) and incubation temperature on fermentation characteristics and microbial communities using the rumen simulation technique. A completely randomized experiment was conducted over a 28-day period using a Rusitec system. The experiment was divided into four periods of 7 days each, the first of which was a 7-day adaptation period followed by three experimental periods. The two treatments consisted of a control diet (no pequi oil inclusion) and a diet supplemented with pequi oil (1.5 mL/day) which increased the dietary fat content to 6% (dry matter, DM-basis). Three fermenter vessels (i.e., replicates) were allocated to each treatment. In the first experimental period, the incubation temperature was maintained at 39°C, decreased to 35°C in the second experimental period and then increased again to 39°C in the third. Pequi oil was continuously supplemented during the experiment. Microbial communities were assessed using high-throughput sequencing of the archaeal and bacterial 16S rRNA gene. Methane production was reduced by 57% following a 4°C decrease in incubation temperature. Supplementation with pequi oil increased the dietary fat content to 6% (DM-basis) but did not affect methane production. Analysis of the microbiota revealed that decreasing incubation temperature to 35°C affected the archaeal and bacterial diversity and richness of liquid-associated microbes, but lipid supplementation did not change microbial diversity.

Tucumã oil is sourced from the fruit pulp of the tucumã tree and contains high concentrations of unsaturated fatty acids and carotenoids. Due to these properties it may have the potential to decrease enteric methane (CH4) from ruminants when included in the diet. The objective of this study was to determine the effect of oil mechanically extracted from the fruit pulp of tucumã on fermentation characteristics, CH4 production and the microbial community using the rumen stimulation technique. Treatments consisted of a control diet (forage:concentrate; 70:30), and tucumã oil included at 0.5 or 1.0% (v/v). Addition of tucumã oil linearly decreased (P < 0.01) dry matter disappearance. Total gas (mL/d) and carbon dioxide (CO2) production (mL/d, mL/g DM) were unaffected (P ≥ 0.36) to increasing addition of tucumã oil where 0.5% (v/v) of Tucumã oil numerically increased both variables. Acetate and butyrate percentages of total VFA were linearly decreased (P ≤ 0.01) and propionate and valerate percentages of total VFA were linearly increased (P < 0.01) by increasing concentrations of tucumã oil added to the substrate. The ratio of acetate to propionate was linearly decreased (P < 0.01) with increasing concentration of tucumã oil. Methane production (mL/d) was linearly decreased (P = 0.04) with increasing addition of tucumã oil to the substrate. Tucumã oil reduced the bacterial richness and diversity when included at 1.0% (v/v) in both solid- and liquid- associated microbes. The abundance of the genera Fibrobacter and Rikenellaceae RC9 gut group were decreased and Pyramidobacter, Megasphaera, Anaerovibrio, and Selenomonas were enriched by the addition of 1.0% tucumã oil. In conclusion, tucumã oil resulted in the favorable shift in fermentation products away from acetate toward propionate, decreasing the production of CH4 when tucumã oil was included at 1.0% (v/v), however, substrate digestibility was also inhibited. The rumen microbiota was also altered by the addition of tucumã oil.

The reduction of methane emissions by ruminants is a highly desirable goal to mitigate greenhouse gas emissions. Various feed additives have already been tested for their ability to decrease methane production; however, practical use is often limited due to negative effects on rumen fermentation or high costs. Organosulphur compounds from garlic (Allium sativum) and flavonoids have been identified as promising plant-derived compounds which are able to reduce methane production. Here, we evaluated the effects of a combination of garlic powder and bitter orange (Citrus aurantium) extracts, Mootral, on ruminal methane production, ruminal fermentation and the community of methanogenic Archaea by using the rumen simulation technique as ex vivo model. The experiment consisted of an equilibration period of 7 days, an experimental period of 8 days and a withdrawal period of 4 days. During the experimental period three fermenters each were either treated as controls (CON), received a low dose of Mootral (LD), a high dose of Mootral (HD), or monensin (MON) as positive control. Application of Mootral strongly reduced the proportion of methane in the fermentation gas and the production rate of methane. Moreover, the experimental mixture induced a dose-dependent increase in the production rate of short chain fatty acids and in the molar proportion of butyrate. Some effects persisted during the withdrawal period. Both, single strand conformation polymorphism and Illumina MiSeq 16S rRNA amplicon sequencing indicated an archaeal community distinct from CON and MON samples in the LD and HD samples. Among archaeal families the percentage of Methanobacteriaceae was reduced during application of both doses of Mootral. Moreover, several significant differences were observed on OTU level among treatment groups and after withdrawal of the additives for LD and HD group. At day 14, 4 OTUs were positively correlated with methane production. In conclusion this mixture of garlic and citrus compounds appears to effectively reduce methane production by alteration of the archaeal community without exhibiting negative side effects on rumen fermentation.

Methane emissions by ruminants contribute to global warming and result in a loss of dietary energy for the animals. One possibility of reducing methane emissions is by dietary strategies. In the present trial, we investigated the long-term effects of Mootral, a feed additive consisting of garlic powder (Allium sativum) and bitter orange extracts (Citrus aurantium), on fermentation parameters and the microbial community in the rumen simulation technique (RUSITEC) system. The experiment lasted 38 days and was divided into three phases: an equilibration period of 7 days, a baseline period (BL) of 3 days, and experimental period (EP) of 28 days. Twelve fermentation vessels were divided into three groups (n = 4): control (CON), short-term (ST), and long-term (LT) application. From day 11 to day 27, 1.7 g of Mootral was added to the ST vessels; LT vessels received 1.7 g of Mootral daily for the entire EP. With the onset of Mootral application, methane production was significantly reduced in both groups until day 18. Thereafter, the production rate returned to the initial quantity. Furthermore, the short chain fatty acid fermentation profile was significantly altered by Mootral application; the molar proportion of acetate decreased, while the proportions of propionate and butyrate increased. Metabolomic analysis revealed further changes in metabolite concentrations associated with the Mootral supplementation period. The methyl coenzyme-M reductase gene copy number was reduced in the liquid and solid phase, whereas the treatment did not affect the abundance of bacteria. At the end of the BL, Methanomicrobia was the most abundant archaeal class. Mootral supplementation induced an increase in the relative abundance of Methanomassiliicoccales and a reduction in the relative abundance of Methanomicrobia, however, this effect was transient. Abundances of bacterial families were only marginally altered by the treatment. In conclusion, Mootral has the transient ability to reduce methane production significantly due to a selective effect on archaea numbers and archaeal community composition with little effect on the bacterial community.