| Summary: | Abstract Background This study was conducted to examine effects of nitrate on ruminal methane production, methanogen abundance, and composition. Six rumen-fistulated Limousin×Jinnan steers were fed diets supplemented with either 0% (0NR), 1% (1NR), or 2% (2NR) nitrate (dry matter basis) regimens in succession. Rumen fluid was taken after two-week adaptation for evaluation of in vitro methane production, methanogen abundance, and composition measurements. Results Results showed that nitrate significantly decreased in vitro ruminal methane production at 6 h, 12 h, and 24 h (P < 0.01; P < 0.01; P = 0.01). The 1NR and 2NR regimens numerically reduced the methanogen population by 4.47% and 25.82% respectively. However, there was no significant difference observed between treatments. The alpha and beta diversity of the methanogen community was not significantly changed by nitrate either. However, the relative abundance of the methanogen genera was greatly changed. Methanosphaera (PL = 0.0033) and Methanimicrococcus (PL = 0.0113) abundance increased linearly commensurate with increasing nitration levels, while Methanoplanus abundance was significantly decreased (PL = 0.0013). The population of Methanoculleus, the least frequently identified genus in this study, exhibited quadratic growth from 0% to 2% when nitrate was added (PQ = 0.0140). Conclusions Correlation analysis found that methane reduction was significantly related to Methanobrevibacter and Methanoplanus abundance, and negatively correlated with Methanosphaera and Methanimicrococcus abundance.
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