Assessment of Potential Anti-Methanogenic and Antimicrobial Activity of Ethyl Nitroacetate, α-Lipoic Acid, Taurine and L-Cysteinesulfinic Acid In Vitro.

Livestock producers need new technologies to maintain the optimal health and well-being of their animals while minimizing the risks of propagating and disseminating pathogenic and antimicrobial-resistant bacteria to humans or other animals. Where possible, these interventions should contribute to the efficiency and profitability of animal production to avoid passing costs on to consumers. In this study, we examined the potential of nitroethane, 3-nitro-1-propionate, ethyl nitroacetate, taurine and L-cysteinesulfinic acid to modulate rumen methane production, a digestive inefficiency that results in the loss of up to 12% of the host's dietary energy intake and a major contributor of methane as a greenhouse gas to the atmosphere. The potential for these compounds to inhibit the foodborne pathogens, Escherichia coli O157:H7 and Salmonella Typhimurium DT104, was also tested. The results from the present study revealed that anaerobically grown O157:H7 and DT104 treated with the methanogenic inhibitor, ethyl nitroacetate, at concentrations of 3 and 9 mM had decreased (p < 0.05) mean specific growth rates of O157:H7 (by 22 to 36%) and of DT104 (by 16 to 26%) when compared to controls (0.823 and 0.886 h-1, respectively). The growth rates of O157:H7 and DT104 were decreased (p < 0.05) from controls by 31 to 73% and by 41 to 78% by α-lipoic acid, which we also found to inhibit in vitro rumen methanogenesis up to 66% (p < 0.05). Ethyl nitroacetate was mainly bacteriostatic, whereas 9 mM α-lipoic acid decreased (p < 0.05) maximal optical densities (measured at 600 nm) of O157:H7 and DT104 by 25 and 42% compared to controls (0.448 and 0.451, respectively). In the present study, the other oxidized nitro and organosulfur compounds were neither antimicrobial nor anti-methanogenic.

Astragallus mollissimus plant extract: a strategy to reduce ruminal methanogenesis.

Ruminal methanogenesis is considered an inefficient process as it can result in the loss of 4 to 12% of the total energy consumed by the ruminant. Recent studies have shown that compounds such as nitroethane, 2-nitroethanol, 2-nitro-1-propanol, and 3-nitro-1-propionic acid are capable of inhibiting methane production during in vitro studies. However, all of these nitrocompounds came from a synthetic origin, which could limit their use. In contrast, some plants of the Astragallus genus produce a natural nitrocompound, although its anti-methanogenic effect has not been evaluated. To determine the anti-methanogenic effect, in vitro cultures of freshly collected mixed populations of ruminal microbes were supplemented with A. mollissimus extracts (MISER). Cultures supplemented with 2-nitroethanol, ethyl 2-nitroacetate, or nitroethane were used as positive controls whereas distilled water was added to the untreated control tubes. After a 24 h incubation period, the methane production was reduced by more than 98% for the samples treated with A. mollissimus extract (P < 0.05) compared to the untreated controls (10.2 ± 0.1 mmol mL-1 incubated liquid). Cultures supplemented with MISER produced a greater (P < 0.05) amount of total VFA, compared to the rest of treated and untreated cultures. Considering that there are significant differences between MISER treatment, positive controls and untreated cultures (P < 0.05) regarding the amounts of total gas, gas composition (CH4 and H2), and the amount of VFA produced, it is concluded that Astragallus mollissimus poses an alternative strategy to reduce ruminal methanogenesis. To further explore such alternative, it is necessary to determine if the metabolization byproducts are safe and/or useful for the animal.

In vitro reduction of methane production by 3-nitro-1-propionic acid is dose-dependent1.

Methanogenesis is a metabolic process that allows the rumen ecosystem the ability to maintain the low hydrogen partial pressures needed for proper digestive function. However, rumen methanogenesis is considered to be an inefficient process because it can result in the loss of 4% to 12% of the total energy consumed by the host. Recent studies have shown that some short-chain nitrocompounds such as nitroethane, 2-nitroethanol, 2-nitro-1-propanol, and 3-nitro-1-propionic acid (3NPA) are capable of inhibiting the production of methane during in vitro culture; nevertheless, optimal supplementation doses have yet to be determined. In the present study, in vitro cultures of freshly collected mixed populations of ruminal microbes were supplemented with the naturally occurring nitrocompound, 3NPA, to achieve 0, 3, 6, 9, or 12 mM. Analysis of fermentation products after 24 h of incubation revealed that methane (CH4) production was reduced in a dose-dependent manner by 29% to 96% (P < 0.05) compared with the amount produced by untreated controls (15.03 ± 0.88 µmol mL-1 incubated liquid). Main effects of the supplement were also observed, which resulted in a reduction (P < 0.05) on amounts of total gas and volatile fatty acids (VFA) produced, as well as in an increase of 0.07 to 0.30 µmol mL-1 on rates of 3NPA degradation. Changes in production of metabolites as CH4, hydrogen (H2), VFA, and NH3 indicated that the fermentation efficiency was not compromised dramatically by 3NPA treatment in moderate doses of 6 and 9 mM. Results further revealed that the metabolism of the 3NPA by microbial populations is also dose-dependent. The microbes were able to metabolize more than 75% of the added nitrocompound, with the greatest degradation rates in cultures treated with 9-mM 3NPA. Finally, from a practical standpoint, and considering the magnitude of CH4 reduction, effect on VFA, and percentage of metabolized supplement, the most efficacious dose for 3NPA administration may be between 3 and 9 mM.

Effect of sole or combined administration of nitrate and 3-nitro-1-propionic acid on fermentation and Salmonella survivability in alfalfa-fed rumen cultures in vitro.

Ruminal methanogenesis is a digestive inefficiency resulting in the loss of dietary energy consumed by the host and contributing to environmental methane emission. Nitrate is being investigated as a feed supplement to reduce rumen methane emissions but safety and efficacy concerns persist. To assess potential synergies of co-administering sub-toxic amounts of nitrate and 3-nitro-1-propionate (NPA) on fermentation and Salmonella survivability with an alfalfa-based diet, ruminal microbes were cultured with additions of 8 or 16mM nitrate, 4 or 12mM NPA or their combinations. All treatments decreased methanogenesis compared to untreated controls but volatile fatty acid production and fermentation of hexose were also decreased. Nitrate was converted to nitrite, which accumulated to levels inhibitory to digestion. Salmonella populations were enriched in nitrate only-treated cultures but not in cultures co- or solely treated with NPA. These results reveal a need for dose optimization to safely reduce methane production with forage-based diets.