3-NOP vs. Halogenated Compound: Methane Production, Ruminal Fermentation and Microbial Community Response in Forage Fed Cattle

Abstract

The aim of this study was to investigate the effects of 3-nitrooxypropanol (3-NOP) and chloroform on methane (CH₄) and H₂ production, ruminal metabolites and microbial community structure in cattle fed a tropical forage diet. Eight rumen-fistulated steers were fed a roughage hay diet (Rhodes grass; Chloris gayana) for 31 days (control period). Four animals received the antimethanogenic compound chloroform (1.6 g chloroform-cyclodextrin/100 kg live weight (LW)) while the other four received 3-NOP (2.5 g 3-NOP/animal/day) for 21 days. Methane decrease compared with control period was similar for both treatments (30-38%) with no differences for expelled H₂ between controls and treatments. Daily weight gain (DWG) was significantly increased when animals were treated with 3-NOP compared with chloroform and control. Regarding the ruminal fermentation parameters increases in ammonia, acetate and branched chain fatty acids were observed with both compounds compared with the controls. Also, methylamines, alcohols and dimethyl sulfone (DMSO₂) concentrations were significantly increased with the treatments compared with control, being greater with 3-NOP. The rumen microbial analyses revealed a similar profile for both treatments, with a shift in operational taxonomic units (OTUs) assigned to the Prevotellaceae and Campylobacteraceae family. Moreover, major archaeal OTUs associated with Methanobrevibacter and Methanosphaera were significantly affected to varying extents based on the inhibitory treatments compared to the control. The abundance of the Methanobrevibacter spp. was decreased by 3-NOP and chloroform, while the Methanomassiliicoccaceae family was inhibited only by 3-NOP. The results suggest that despite the specific mode of action of 3-NOP on methanogens, inhibition of methanogenesis by both compounds resulted in similar responses in metabolism and microbial community structure in the rumen. We hypothesized that these changes were driven by the redirection of metabolic hydrogen ([H]) by both treatments. Therefore results from previous publications using chloroform as an inhibitor of methanogenesis may be useful in predicting ruminal microbiota and fermentation responses to 3-NOP.

Keywords: 3-NOP; NMR; chloroform; methane; methyl compounds; microbiota; rumen.

FIGURE 1

Operational taxonomic units (OTUs) significantly different (q ≤ 0.1 FDR) between 3-NOP treated-steers and control period. Upper axis represents OTU’s with a log2 fold positive difference for 3-NOP treatment relative to control while the lower y-axis is the negative fold difference of the 3-NOP relative to control. Each point represents a single OTU colored by phylum and grouped on the x-axis by taxonomic family level, size of point reflects the log2 mean abundance of the sequence data.

FIGURE 2

OTUs significantly different (q ≤ 0.1 FDR) between chloroform treated-steers and control period. Upper axis represents OTU’s with a log2 fold positive difference for chloroform treatment relative to control while the lower y-axis is the negative fold difference of the chloroform relative to control. Each point represents a single OTU colored by phylum and grouped on the x-axis by taxonomic family level, size of point reflects the log2 mean abundance of the sequence data.

FIGURE 3

OTUs significantly different (q < 0.1 FDR) between 3-NOP and chloroform treated-steers. Upper axis represents OTU’s with a log2 fold positive difference for 3-NOP treatment relative to chloroform while the lower y-axis is the negative fold difference of the 3-NOP relative to chloroform. Each point represents a single OTU colored by phylum and grouped on the x-axis by taxonomic family level, size of point reflects the log2 mean abundance of the sequence data.

FIGURE 4

Quantitative PCR (qPCR) analysis of mcrA gene (methanogens), Methanobrevibacter spp. and Methanomassiliicoccaceae family population changes in response to chloroform or 3-NOP. Asterisks (^∗, ^∗∗, ^∗∗∗) denote significant differences of treatment compared to control periods (P < 0.05), (P < 0.01) and (P < 0.01), respectively; the letter (t) denote a trend between control and treatment (P < 0.1). The y-axis denotes fold change from control period.