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.