Can adaptation to nitrate supplementation and provision of fermentable energy reduce nitrite accumulation in rumen contents in vitro?

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Nitrate (NO3–) supplementation is a promising methane mitigation strategy for ruminants, but can cause nitrite (NO2–) poisoning. Because some nitrite reductases are NADH-dependent, we hypothesised that replacing glucose with glycerol would increase the NADH yield and so enhance nitrite reductase activity and reduce ruminal NO2– accumulation and toxicity risk. We also hypothesised that adapting sheep to dietary NO3– would limit the accumulation of NO2– when NO3– was added to rumen fluid. Changes in NO3– and NO2– catabolism and CH4 production, resulting from supplementation with glycerol to enhance NADH supply, were studied in vitro. In Experiment 1, rumen fluid from sheep adapted to dietary NO3– (2% of DM intake) or urea (1.1% of DM intake) was incubated with NO3– or urea, respectively. Additionally, ground oaten hay was added to incubations alone (control), or with glucose or glycerol. In Experiement 2, sheep were adapted for 9 weeks to dietary NO3– or urea. Nitrate (2% NO3– of substrate DM) was added to incubated digesta from NO3–- or urea-supplemented sheep, while urea (1.1% of substrate DM) was added to digesta from urea-supplemented sheep. In both studies, triplicate incubations were terminated at nine time points up to 24 h. Methane emissions were lower in all NO3– treatments (P < 0.05). Contrary to our hypotheses, both glycerol supplementation (Experiment 1) and prior adaptation to NO3– (Experiment 2) increased NO2– accumulation. In Experiment 1, there was no difference in ruminal NO2– concentration between the unsupplemented control and added glucose treatments. Nitrous oxide accumulated in NO3– treatments only with rumen fluid from sheep adapted to dietary urea (P < 0.05). In summary, NO2– accumulation in vitro was not reduced by adaptation to NO3– or by glucose or glycerol supplementation, disproving the hypotheses regarding the role of NADH availability and of NO2– adaptation in reducing ruminal NO2– accumulation and toxicity risk.