Dietary nitrate and presence of protozoa increase nitrate and nitrite reduction in the rumen of sheep

Nitrate (NO₃^–) supplementation is an effective methane (CH₄) mitigation strategy for ruminants but may produce nitrite (NO₂^–) toxicity. It has been reported that rumen protozoa have greater ability for NO₃^– and NO₂^– reduction than bacteria. It was hypothesised that the absence of ruminal protozoa in sheep may lead to higher NO₂^– accumulation in the rumen and a higher blood methaemoglobin (MetHb) concentration. An in vivo experiment was conducted with defaunated (DEF) and faunated (FAU) sheep supplemented with 1.8% NO₃^– in DM. The effects of rumen protozoa on concentrations of plasma and ruminal NO₃^– and NO₂^–, blood MetHb, ruminal volatile fatty acid (VFA) and ruminal ammonia (NH₃) were investigated. Subsequently, two in vitro experiments were conducted to determine the contribution of protozoa to NO₃^– and NO₂^– reduction rates in DEF and FAU whole rumen digesta (WRD) and its liquid (LIQ) and solid (SOL) fractions, incubated alone (CON), with the addition of NO₃^– or with the addition of NO₂^–. The results from the in vivo experiment showed no differences in total VFA concentrations, although ruminal NH₃ was greater (<i>p</i> < .01) in FAU sheep. Ruminal NO₃^–, NO₂^– and plasma NO₂^– concentrations tended to increase (<i>p</i> < .10) 1.5 hr after feeding in FAU relative to DEF sheep. In vitro results showed that NO₃^– reduction to NH₃ was stimulated (<i>p</i> < .01) by incoming NO₃^– in both DEF and FAU relative to CON digesta. However, adding NO₃^– increased (<i>p</i> < .05) the rate of NO₂^– accumulation in the SOL fraction of DEF relative to both fractions of FAU digesta. Results observed in vivo and in vitro suggest that NO₃^– and NO₂^– are more rapidly metabolised in the presence of rumen protozoa. Defaunated sheep may have an increased risk of NO₂^– poisoning due to NO₂^– accumulation in the rumen.