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

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.