Methane reduction by quercetin, tannic and salicylic acids: influence of molecular structures on methane formation and fermentation in vitro.

Plant secondary metabolites (PSMs) can potentially reduce ruminal methane formation. However, related to differences in their molecular structures, it is not yet clear what causes an anti-methanogenic effect. In an in vitro system simulating rumen fermentation, we investigated the impact of eight compounds with distinct chemical characteristics (gallic and salicylic acids, tannic acid, catechin, epicatechin, quercetin, rutin, and salicin) when added to a basal feed (maize silage) at a concentration of 12% of the feed dry matter. After 48 h of incubation in buffered rumen fluid, methane production was significantly lowered by quercetin (43%), tannic acid (39%) and salicylic acid (34%) compared to the control (maize silage alone) and without changes in total volatile fatty acid production during fermentation. No other PSM reduced methane formation as compared to control but induced significant differences on total volatile fatty acid production. The observed differences were related to lipophilicity, the presence of double bond and carbonyl group, sugar moieties, and polymerization of the compounds. Our results indicate the importance of distinct molecular structures of PSMs and chemical characteristics for methane lowering properties and volatile fatty acid formation. Further systematic screening studies to establish the structure-function relationship between PSMs and methane reduction are warranted.

Changes in the Metagenome-Encoded CAZymes of the Rumen Microbiome Are Linked to Feed-Induced Reductions in Methane Emission From Holstein Cows.

Enteric methane (CH4) emission from cattle is strongly linked to the feeding regime and the rumen microbial community structure. Here, we report that feed-induced CH4-reducing effects correlate with specific alterations in the profile of the microbiome-encoded carbohydrate-active enzymes predicted from the rumen fluid metagenome. Rumen microbiome samples were obtained by mouth-tube sampling from 12 lactating Holstein cows after 3-4 weeks of feeding with three different concentrate-to-forage-ratio diets, i.e., standard, high, and extremely high levels of concentrate (4 cows per group; constant dry matter intake in the three groups). Increased inclusion of concentrate involved increased starch levels in the diet at the expense of fiber. The extreme diet resulted in 48% reduction of the CH4 emission per kg dry matter intake compared to the standard diet. From metagenome sequencing of the rumen fluid samples from each cow, 561 different microbial strains (bins) could be derived from analysis of 260 billion DNA base pairs. In the cows fed, the extreme diet, the relative abundance of the majority of the bins, was significantly altered compared to the other groups. Fibrobacterota and Verrucomicrobiota were less abundant in the Extreme group. Surprisingly, no significant abundance changes were observed among Archaea and Bacteroidota, although abundance changes of individual bins of these phyla were found. For each of the 561 bins, the functions of the metagenome-encoded carbohydrate-active enzymes were predicted by bioinformatics using conserved unique peptide pattern (CUPP) analysis. By linking each of the predicted molecular functions of the enzymes to their substrates, changes were found in the predicted abundance of the different enzyme types. Notably, the decreased CH4 emission of the extreme diet group was concurrent with a profound decrease in the xylan-active enzymes, targeting the xylan backbone ß-1,4-linkages, acetyl-, feruloyl-, and methyl-glucuronoyl substitutions in xylan. This work provides a first enzyme-conversion-based characterization of how extreme feeding, i.e., lowered forage, can drive rumen microbiome changes that support decreased CH4 emission via a changed carbohydrate-active enzyme profile. The data, furthermore, provide a metagenome-wide catalog of enzymes, underpinning the microbial conversion of different feed fibers (the enzymes attacking specific carbohydrate linkages) in the rumen of Holstein cows.

Untargeted Metabolomics Combined with Solid Phase Fractionation for Systematic Characterization of Bioactive Compounds in Hemp with Methane Mitigation Potential.

This study systematically evaluates the presence of methane mitigating metabolites in two hemp (Cannabis sativa L.) varieties, Futura 75 and Finola. Hemp metabolites were extracted with methanol and fractionated using Solid Phase Extraction (SPE). Extracts, fractions, and the remaining pulp were screened for their methane mitigating potential using an in vitro model of rumen fermentation. The bioactive metabolites were identified with Liquid Chromatography-Mass Spectrometry (LC-MS). When incubated with a standard feed (maize silage), the extract of Futura 75 significantly reduced methane production compared to that of control (without added extract) and without negative effects on feed degradability and volatile fatty acid patterns. The compounds responsible for the methane mitigating effect were assigned to flavonoid glycosides. However, none of the fractions of Futura 75 or the pulp exhibited similar effect on methane emission. Butyric acid concentration in the fermentation inoculum was significantly increased, which could indicate why methane production was higher, when incubated with the fractions and the pulp. The extract of Finola did not show a similar significant effect, however, there was a numerical tendency towards lower methane production. The difference in methane mitigating properties between Cannabis sativa L. Futura 75 and Finola, could be related to the content of bioactive flavonoids.

Rumen and Fecal Microbial Community Structure of Holstein and Jersey Dairy Cows as Affected by Breed, Diet, and Residual Feed Intake.

Identifying factors that influence the composition of the microbial population in the digestive system of dairy cattle will be key in regulating these populations to reduce greenhouse gas emissions. In this study, we analyzed rumen and fecal samples from five high residual feed intake (RFI) Holstein cows, five low RFI Holstein cows, five high RFI Jersey cows and five low RFI Jersey cows, fed either a high-concentrate diet (expected to reduce methane emission) or a high-forage diet. Bacterial communities from both the rumen and feces were profiled using Illumina sequencing on the 16S rRNA gene. Rumen archaeal communities were profiled using Terminal-Restriction Fragment Length Polymorphism (T-RFLP) targeting the mcrA gene. The rumen methanogen community was influenced by breed but not by diet or RFI. The rumen bacterial community was influenced by breed and diet but not by RFI. The fecal bacterial community was influenced by individual animal variation and, to a lesser extent, by breed and diet but not by RFI. Only the bacterial community correlated with methane production. Community differences seen in the rumen were reduced or absent in feces, except in the case of animal-to-animal variation, where differences were more pronounced. The two cattle breeds had different levels of response to the dietary intervention; therefore, it may be appropriate to individually tailor methane reduction strategies to each cattle breed.