Ruminal fermentation of anti-methanogenic nitrate- and nitro-containing forages in vitro

Authors: Anderson, Robin; Ripley, Laura; BOWMAN, JAN - Montana State University; Callaway, Todd; Genovese, Kenneth - Ken; Beier, Ross; Harvey, Roger; Nisbet, David

Submitted to: Frontiers in Veterinary Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/28/2016
Publication Date: 8/11/2016
Publication URL: http://handle.nal.usda.gov/10113/63136
Citation: Anderson, R.C., Ripley, L.H., Bowman, J.G., Callaway, T.R., Genovese, K.J., Beier, R.C., Harvey, R.B., Nisbet, D.J. 2016. Ruminal fermentation of anti-methanogenic nitrate- and nitro-containing forages in vitro. Frontiers in Veterinary Science. 3:62. doi: 10.3389/fvets.2016.00062.

Interpretive Summary: Methane production by microbes in the gut of cows, sheep, and goats is an inefficient digestive process that results in the loss of as much as 16% of the dietary energy consumed by the animal and contributes nearly 20% of the United States' emission of this greenhouse gas. The chemicals nitrate, 3-nitro-1-propionic acid (NPA), and 3-nitro-1-propanol (NPOH) are recognized as potent inhibitors of methane production by microbes in the gut of cows, sheep, and goats and thus have the potential to reduce economic and environmental costs associated with this digestive inefficiency. These chemicals can accumulate naturally in forages that can be fed to animals, and thus can possibly be part of a green technology to reduce methane production. However, if fed at too high an amount, the forages containing these chemicals can potentially be poisonous to animals, and thus research is needed to learn how to safely use these forages. In the present experiment, we found that nitrate-, NPA-, or NPOH- containing forages effectively decreased methane production by populations of bacteria from the gut of cows. Methane-producing bacterial populations were inhibited most effectively with the NPA- and NPOH-containing forages. Hydrogen, an end product of forage digestion, accumulated in bacterial populations incubated with forages containing nitrate, NPA, or NPOH but was dramatically higher in populations incubated with the NPA-containing forage. This finding provides evidence of a specific mechanism of action for NPA, implicating its effects on a specific hydrogen-using enzyme within the bacterial population. Accumulations of other endproducts revealed compensatory adaptations by the microbial populations incubated with the nitrate-containing and NPA- and NPOH-containing forages, which provides further evidence of how these chemicals affect the microbial population. These results demonstrate that forages containing nitrate, NPA, and NPOH can be used to inhibit methanogenesis and provide important information on the mechanism of action of the anti-methane chemicals in these forages. Ultimately, this research can be used to develop feeding strategies that help farmers and ranchers increase production of safe and wholesome meat and milk at lower cost, while at the same time decreasing the global carbon footprint of American agriculture.

Technical Abstract: Nitrate, 3-nitro-1-propionic acid (NPA), and 3-nitro-1-propanol (NPOH) can accumulate in forages and be poisonous to animals if fed at high enough amounts. These chemicals are also recognized as potent anti-methanogenic compounds, but plants naturally containing these chemicals have been studied little in this regard. Presently, we found that nitrate-, NPA-, or NPOH- containing forages effectively decreased methane production, by 34 to 98%, during in vitro fermentation by mixed populations of ruminal microbes compared to fermentation by populations incubated similarly with alfalfa. Methane production was further decreased, although not necessarily significantly, during incubation of populations inoculated with Denitrobacterium detoxificans, a ruminal bacterium known to reductively metabolize nitrate, NPA, and NPOH. Inhibition of methanogen populations was greatest with the NPA- and NPOH-containing forages. Hydrogen accumulated in all ruminal populations incubated with forages containing nitrate, NPA, or NPOH, but was dramatically higher, exceeding 40 µmol hydrogen/mL, in non D. detoxificans-inoculated populations incubated with NPA-containing milkvetch. This possibly reflects the inhibition of hydrogenase-catalyzed uptake of hydrogen produced via conversion of the added 50 µmol formate/mL to hydrogen. Accumulations of volatile fatty acids revealed compensatory changes in fermentation in the nitrate-containing and NPA- and NPOH-containing forages as evidenced by lower accumulations of acetate, and in some cases higher accumulations of butyrate. Accumulations of ammonia, iso-buytrate, and in some cases iso-valerate, were also lower in incubations with nitrate-, NPA-, and NPOH-containing forages than with alfalfa, thus indicating a potential alteration of amino acid fermentation. Results reveal that nitrate, NPA, and NPOH that accumulate naturally in forages can be made available with ruminal incubations to inhibit methanogenesis. Further research is needed to determine if diets can be formulated with nitrate-, NPA-, and NPOH-containing forages to achieve efficacious mitigation in ruminant methane emissions without adversely affecting fermentative efficiency.