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.
Paraffin-wax-coated nitrate salt inhibits short-term methane production in sheep and reduces the risk of nitrite toxicity
Inclusion of nitrate in the diet of ruminants has been effective in reducing enteric emission of the greenhouse gas methane, but increases the risk of nitrate toxicity in the animal. An experiment was conducted to investigate if coating nitrate salts with lipid would reduce risks of nitrite toxicity in sheep without compromising the methane mitigating effect achieved using uncoated nitrate. Three forms of nitrate (uncoated nitrate; coated with palm oil or coated with paraffin wax) were administered intraruminally to sheep, with nitrate toxicity risk being evaluated by determining blood methaemoglobin (MetHb) levels. Nitrate and nitrite concentrations in plasma and rumen fluid, as well as methane and nitrous oxide production were also evaluated. Sheep supplemented with isonitrogenous amounts of urea were used as negative controls. There was no significant effect of palm oil coating on MetHb but coating with paraffin wax lowered MetHb levels, rumen and plasma nitrate concentrations (P < 0.05) relative to concentrations in urea-supplemented sheep. Total VFA concentrations in rumen fluid were unaffected by coating nitrate, but acetate proportion increased while butyrate and propionate proportions declined over time in all treatments after intraruminal nitrate administration (P < 0.05). It is suggested that these changes were caused by the strong capacity of nitrate to act as an electron acceptor. There was substantial variation between animals in ruminal nitrate and nitrite concentrations and in blood MetHb when the same mass of nitrate was administered directly into the rumen, showing that individuals differ in their ability to metabolize nitrate. Whereas methane production over the 22 h period of measurement was unaffected by the treatments, methane production during the first 3 h of measurement post-feeding was reduced similarly by both coated and uncoated nitrate supplements compared to urea. The small amount of supplemented nitrate introduced and the rapidity of nitrate reduction may both explain why methane mitigation was only observed for a short period after administering the treatments. Over 22 h in respiration chambers, nitrous oxide emissions were significantly increased by uncoated nitrate supplements compared to urea (P < 0.05). Nitrous oxide emissions by sheep fed coated nitrate did not differ from those of sheep fed urea. It is concluded that coating dietary nitrate can protect sheep against nitrite toxicity without adversely affecting methane mitigation.