PSIV-2 Effect of Select Nitrocompounds on Rumen Methane Inhibition and Antimicrobial Activity in Vitro
Authors: Corona Alejandra M , Garza Sarina A , Anderson Robin C , Fonseca Mozart A , Kotzur Jackie , Mast Natasha L 
Published: November 2023

Abstract
Ruminal methane production accounts for losses of 4 to 12% of the gross energy intake of an animal and contributes nearly 25% of U.S. emissions of this greenhouse gas. Certain short-chain nitrocompounds inhibit ruminal methane production, which may yield opportunities for producers to reduce economic and environmental costs associated with methane emission. Accordingly, the methane-inhibiting potential of several nitrocompounds were compared while evaluating their potential to inhibit indigenous coliforms and experimentally inoculated Salmonella typhimurium, to achieve 103 cells/mL, within populations of ruminal microbes. Rumen fluid collected from a cannulated forage-fed steer was used to inoculate (1 mL/tube, 3 tubes/treatment) an initial series of culture tubes preloaded without or with nitroethane, 2-nitroethanol, ethyl nitroacetate or ethyl-2-nitropropionate (each at 9 mM), 0.2 g ground alfalfa and 9 mL of a basal rumen fluid-based medium prepared under a 50:50 H2:CO2 atmosphere. Tubes were incubated at 39oC for 24-h. Upon completion of this first incubation series, 1 mL was withdrawn from each cultured tube and used to inoculate a second series of tubes prepared with fresh medium and treatments and incubated 24-h as above. An analysis of variance revealed that 24-h methane accumulations were reduced (P &lt; 0.05) by 99% in all nitrocompound-treated incubations compared with accumulations in non-treated controls (19.2 ± 2.9 and 12.3 ± 1.2 µmol/mL; mean ± SD for the first and second series, respectively). The decreased (P &lt; 0.05) accumulation of methane in the controls during the second incubation series likely reflects diminished protozoal populations that would be expected during in vitro incubations. Main effects of treatment or treatment by series interactions were not observed (P &gt; 0.05) on accumulations of hydrogen above the 38 µmol/mL initially present in the 50% H2:CO2 headspace. However, a main effect of series was observed (P &lt; 0.05), with more hydrogen accumulating during the first than during the second incubation series (14.7 ± 10.4 versus 0.9 ± 6.4 µmol H2/mL, respectively) thus implicating a diminution in H2-producing microbes. Selective enumeration of wildtype coliforms (on 3M Petrifilm) from samples collected at intervals during the first series revealed 0.6 to 1.2 log10 unit growth inhibition (P &lt; 0.05) by 2-nitroethanol, ethyl nitroacetate and ethyl-2-nitroacetate after 6 h and by 2-nitroethanol and ethyl nitroacetate after 24-h incubation of the rumen cultures, with controls achieving 4.83 ± 0.7 and 3.93 ± 0.3 log10 colony forming units (CFU)/mL after 6 and 24-h, respectively. Antimicrobial activity of the nitrocompounds against S. typhimurium, enumerated on Brilliant Green agar, was not observed, with counts averaging 3.3 ± 0.3 and 3.0 ± 0.3 log10 CFU/mL after 6 and 24-h incubation, respectively. These results confirm the tested nitrocompounds exhibit potent methane-inhibiting activity but only modest bacteriostatic activity.