Supplementing ruminants with dietary nitrate (NO₃) is an effective methane mitigation strategy if it can be managed so as to not expose ruminants to any risk of clinical nitrite (NO₂) toxicity. The objective of this thesis was firstly to deepen the understanding for NO₃ metabolism in sheep and secondly to develop practical strategies to reducing risk of NO₂ toxicity in sheep supplemented with dietary NO₃.

It has been previously established, that in the rumen NO₃ is reduced to NO₂ and then to NH₃, and that supplementing with excessive amounts of NO₃ can expose ruminants to NO₂ toxicity due to the absorption of NO₂. This thesis reports a series of five investigations of NO₃ metabolism by sheep and identifies:

Nitrate, like urea, is ‘recycled’ within the ruminant. Transfer of ruminal ¹⁵NO₃^--N into the blood and transfer of blood NO₂-N into the rumen being quantified. Only 20% of rumen NO₃^-and 30% of blood NO₂^- were recovered in urine.

That in hourly fed sheep approximately 90% of dietary NO₃^- was rapidly converted to NH₃ in the rumen, with the remainder leaving the rumen by absorption into the bloodstream or passage to the lower gastro-intestinal tract.

Within the rumen, the conversion of NO₃^-to NH₃ is neither simple nor complete. In vitro and in-vivo studies showed NO₃^-is reduced to gaseous nitrous oxide (N₂O) and N₂O may be further metabolised to N₂ gas by the rumen microbiota. Approximately 0.04% and 3.0% of dosed NO₃^--N was recovered over 10 h from sheep as N₂O and N₂ respectively, and this was not affected by whether sheep had prior adaption to NO₃^- or not, identifying denitrification as a reaction not previously reported from the rumen.

From this understanding and a review of the literature on ruminant NO₃ metabolism, eight critical control points for reducing the risk of nitrite toxicity (methaemoglobinaemia), were identified and the potential for manipulating five of these evaluated.

Reducing the rate at which NO₃ became available to the rumen biota by coating calcium nitrate with paraffin wax significantly reduced blood methaemoglobin level (MetHb; an indicator of NO₂ toxicity) in sheep supplemented with NO₃.

The extent of methaemoglobinaemia could also be reduced by the daily ration being consumed at shorter intervals rather than in a single bout, and this established that feed management is pivotal to safe feeding of NO₃^-containing diets.

Enhancing the rumen’s capacity to reduce potentially toxic NO₂ ^-by supplying Propionibactericum acidicpropionici as a direct fed microbial was ineffective in reducing blood MetHb or NO₂^-concentration of sheep fed NO₃^- supplemented diets.

Attempts to increase the rate of removal of NO₂^-from the rumen by providing a substrate (glycerol) to stimulate NADH availability in the rumen, and accelerate the nitrite reductase enzyme system did not reduce the concentration of NO₂ in incubations of rumen contents supplemented with NO₃^-.

We found no evidence that adapting sheep to dietary NO₃^- protected them against NO₂^- toxicity. Indeed, in vitro more NO₂^- accumulated in incubation when donors where adapted to dietary NO₃^-. Also, no signs of reduced MetHb were noticed after several weeks of NO₃^-supplementation in vivo.

Other critical control points such as regulating microbial uptake of NO₃ and ruminal absorption of NO₃ and NO₂ were unable to be assessed in this thesis.

The studies reported here also confirmed the practical impacts of NO₃ as an effective supplement for reducing enteric methane emissions and increasing wool growth of sheep. As well as providing a better understanding of NO₃^-metabolism, studies also showed that the greenhouse gas (GHG) abatement impact of methane mitigation may be partly offset by an associated production of the potent GHG, N₂O. Discovery of the production of N₂O and N₂ from NO₃^-in the rumen and identification of recycling of blood NO₂^- to the rumen has expanded our understanding of NO₃^-metabolism. Coating NO₃^-to decrease the rapidity of NO₃^- release in the rumen as a strategy to reduce NO₂ toxicity was effective but needs further investigation. The applicability of feed grade NO₃^-as a commercially available feed additive will also depend on the cost of NO₃ and the additional cost of the technology to ensure its safe feeding, compared to the cheaper alternative non-protein nitrogen source, urea.

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.

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.