Batch culture analysis to identify potent organic acids for suppressing ruminal methane production.

We performed an in vitro rumen batch culture study to screen 11 commercially available organic acids for methane-suppressing ability and analyzed the rumen microbiota to determine the mode of action of the acids that showed potent methane-suppressing activity. Nine of the 11 acids showed methane-suppressing activity. Maleic anhydride, itaconate, citrate, and fumarate, which showed the highest activity, were further examined. These four acids showed methane-suppressing activity irrespective of the hay-to-concentrate ratios of the substrate. Maleic anhydride and itaconate decreased total gas and short-chain fatty acid production. Maleic anhydride and fumarate increased propionate production, while itaconate increased butyrate production. Maleic anhydride, itaconate, and citrate increased lactate production. Fumarate increased the abundance of bacteria involved in propionate production. Maleic anhydride, itaconate, and citrate increased the abundance of bacteria involved in lactate production. Thus, the results indicate that maleic anhydride, itaconate, and citrate may decrease methane in part by stimulating the acrylate pathway.

Conditions stimulating neutral detergent fiber degradation by dosing branched-chain volatile fatty acids. I: Comparison with branched-chain amino acids and forage source in ruminal batch cultures.

Three experiments assessed branched-chain volatile fatty acid (BCVFA) stimulation of neutral detergent fiber (NDF) disappearance after 24 h of incubation in batch cultures derived from ruminal fluid inocula that were enriched with particulate-phase bacteria. In experiment 1, a control was compared with 3 treatments with isomolar doses of all 3 BCVFA (plus valerate), all 3 branched-chain AA (BCAA), or half of each BCVFA and BCAA mix with either alfalfa or grass hays (50%) and ground corn grain (50%). A portion of the BCAA and BCVFA doses were enriched with 13C, and valerate (also enriched with 13C) was added with BCVFA. Although BCAA yielded a similar production of BCVFA compared with dosing BCVFA, equimolar substitution of BCVFA for BCAA decreased the percentage of N in bacterial pellets when alfalfa hay was fed but increased N when grass hay was fed. Substituting BCVFA for BCAA increased total fatty acid (FA) concentration with alfalfa hay. Dosing of BCAA or BCVFA did not affect total branched-chain FA, iso-FA, or anteiso-FA percentages in bacterial total FA, whereas numerous individual FA isomers and their 13C enrichments were affected by these treatments. Increasing recovery of the 13C dose from respective labeled BCVFA primers indicated facilitated BCVFA uptake and incorporation into FA compared with BCAA, whereas increased recovery of 13C from labeled BCAA in the bacteria pellet but not in the FA fraction suggested direct assimilation into bacterial protein. The BCVFA and valerate were dosed in varying combinations that either summed to 4 mM (experiment 2) or had only 1 mM no matter what combination (experiment 3). In general, grass hay was more responsive to stimulation in NDF digestibility by BCVFA than was alfalfa hay, which was attributed to the higher degradable protein in the latter. The net production of the BCVFA (after subtracting dose) was affected by source and combination of BCVFA. Isovalerate dosing tended to increase its own net production; in contrast, isobutyrate seemed to be used more when it was added alone, but 2-methylbutyrate seemed to be preferred over isobutyrate when 2-methylbutyrate was added. Results supported potential interactions, including potential feedback in production from feed BCAA or increased concentration-dependent competition for dosed BCVFA into cellular products. Under our conditions, the BCVFA appear to be more readily available than BCAA, probably because of regulated BCAA transport and metabolism. Valerate consistently provided no benefit. Using nonparametric ranking, all 3 BCVFA or either isovalerate or isobutyrate (both yielding iso-FA) should be combined with 2-methylbutyrate (yielding anteiso-FA) as a potential opportunity to improve NDF digestibility when rumen-degraded BCAA are limited in diets to decrease environmental impact from N in waste.

In Vitro Gas Production from Batch Cultures of Stomach and Hindgut Digesta of Horses Adapted to a Prebiotic Dose of Fructooligosaccharides and Inulin.

Fructooligosaccharides (FOS) and inulin may modulate hindgut fermentation. It was tested if digesta batch cultures taken from horses adapted to FOS and inulin show different fermentation compared with such taken from nonsupplemented horses. Six horses received 0.15 g FOS and inulin/kg body weight/d via Jerusalem artichoke meal (JAM) upon a hay-based diet; six horses received corncob meal without grains (CMG) as placebo. The horses were euthanized after 20 days. Digesta samples were taken from stomach, cecum, ventral colon ascendens (VCA), and colon transversum (CT). Digesta batch cultures were incubated 48 hours to measure in vitro gas production as well as pre- and post-incubation pH and oxidation-reduction potential (ORP). A distinct fermentation of the surplus of fructans present in the inoculum was found with JAM-adapted batch cultures. Gas production was accelerated in inoculated gastric contents of horses adapted to JAM compared with CMG adapted ones (7.8 vs. 16.4 hours to achieve half of the 48 hours gas quantity, respectively; P > .05). Although buffered, pH decreased during fermentation. Postincubation pH was lower with JAM than CMG-adapted batch cultures (P > .05). Preinoculation ORP was lower with stomach batch cultures adapted to CMG than with such adapted to JAM. The ORP increased twofold from pre- to post-incubation with the latter. Asymptotic maximal gas production decreased gradually using cecum, VCA, or CT digesta. Parts of FOS and inulin of digesta are fermented in the stomach, which reduce possible effects on hindgut fermentation. Elevated fermentation may considerably impact stomach health.

Quantitative evaluation of ruminal methane and carbon dioxide formation from formate through C-13 stable isotope analysis in a batch culture system.

Methane produced from formate is one of the important methanogensis pathways in the rumen. However, quantitative information of CH4 production from formate has been rarely reported. The aim of this study was to characterize the conversion rate (CR) of formic acid into CH4 and CO2 by rumen microorganisms. Ground lucerne hay was incubated with buffered ruminal fluid for 6, 12, 24 and 48 h. Before the incubation, 13C-labeled H13COOH was also supplied into the incubation bottle at a dose of 0, 1.5, 2.2 or 2.9 mg/g of DM substrate. There were no interactions (P>0.05) between dose and incubation time for all variables evaluated. When expressed as an absolute amount (ml in gas sample) or a relative CR (%), both 13CH4 and 13CO2 production quadratically increased (P<0.01) with the addition of H13COOH. The total 13C (13CH4 and 13CO2) CR was also quadratically increased (P<0.01) when H13COOH was added. Moreover, formate addition linearly decreased (P<0.031) the concentrations of NH3-N, total and individual volatile fatty acids (acetate, propionate and butyrate), and quadratically decreased (P<0.014) the populations of protozoa, total methanogens, Methanosphaera stadtmanae, Methanobrevibacter ruminantium M1, Methanobrevibacter smithii and Methanosarcina barkeri. In summary, formate affects ruminal fermentation and methanogenesis, as well as the rumen microbiome, in particular microorganisms which are directly or indirectly involved in ruminal methanogenesis. This study provides quantitative verification for the rapid dissimilation of formate into CH4 and CO2 by rumen microorganisms.

Short communication: A nanoemulsified form of oil blends positively affects the fatty acid proportion in ruminal batch cultures.

Two consecutive rumen batch cultures were used to study the effect of nanoemulsified oils as a new type of supplement, on the in vitro fatty acid proportion and vaccenic acid formation. Three levels (3, 5, and 7%) of 2 different oil blends [soybean:fish oil (SF) or rapeseed-fish oil (RF)] were used. Both oil blends were used either in the raw form (SF or RF, respectively) or in the nanoemulsified form (NSF or NRF, respectively). The diets were the control (0%), which consisted of a dry total mixed ration without any supplements, the control plus 3, 5, or 7% of the SF or RF oil blend in appropriate form (raw or nanoemulsified). For each treatment, 6 incubation vessels were used. Each batch culture was incubated for 24h and conducted twice in 2 consecutive days. All supplements were calculated as a percentage of the substrate dry matter (400mg). Nanoemulsified supplements were recalculated to make sure the oil amount was equal to the raw oil supplementation levels. The results from both experiments indicated that the proportions of vaccenic acid and cis-9,trans-11 C18:2 increased when a raw oil blend was supplemented; on the other hand, no influence of nanoemulsified form of oil blend was observed on the proportion cis-9,trans-11 C18:2. Generally, supplementation with the nanoemulsified oil blends had less effect on biohydrogenation intermediates than the raw form of oil blends. However, the nanoemulsified form had a greater effect on the increase of n-3 and n-6 fatty acids. Nanoemulsified oil blends had a positive effect on decreasing the transformation rate of polyunsaturated fatty acids to saturated fatty acids in the biohydrogenation environment. Supplements of nanoemulsified oil blends tended to be more effective than supplements of raw oils in preserving a greater proportion of polyunsaturated fatty acids in the fermentation culture.