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

Abstract

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 (p < .01) in FAU sheep. Ruminal NO3- , NO2- and plasma NO2- concentrations tended to increase (p < .10) 1.5 hr after feeding in FAU relative to DEF sheep. In vitro results showed that NO3- reduction to NH3 was stimulated (p < .01) by incoming NO3- in both DEF and FAU relative to CON digesta. However, adding NO3- increased (p < .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.

Rumen protozoa are a hub for diverse hydrogenotrophic functions

“…In the rumen, metabolic interactions between ciliate protozoa and nitrate/nitrite reducing bacteria have been proposed. When co-cultured with bacteria, rumen protozoa were reported to accelerate nitrate reduction 11 , and the protozoal fraction, which likely included both protozoa associated prokaryotes showed a greater ability to reduce nitrate without accumulation of nitrite. The possibility that, combined nitrate reduction to nitrite by protozoa, for which genomic evidence exists, with production of hydrogen may lead to the enrichment of nitrite reducing bacteria 52,53 .…”

“…In addition to their still elusive ecological dynamics, ciliate protozoa have also been suggested to have many metabolic and physiological functions in the rumen, most of which not yet elucidated 10 . Some of these functions are further suggested to be a direct consequence of their interactions with the prokaryotic community 11,12 . The most studied functional interaction is their role in enhancing methane production via interspecies hydrogen transfer to methanogenic archaea 12 .…”

Dynamics of the ruminal microbial ecosystem, and inhibition of methanogenesis and propiogenesis in response to nitrate feeding to Holstein calves

“…Ciliated protozoa populations increased significantly as a result of feeding the NIT diet, which is in line with the results obtained in goats ( Zhang et al., 2019 ) and in vitro studies ( Lin et al., 2011 ). Furthermore, in a recent in vivo experiment with defaunated and faunated sheep supplemented with 1.8% of nitrate (on a DM basis), it was reported that nitrate metabolizes more rapidly in the presence of ruminal protozoa, and defaunated sheep may have an increased risk of poisoning due to nitrite accumulation in the rumen ( Villar et al., 2020 ). In contrast, Iwamoto et al.…”

— Invited Review — Ruminal ciliates as modulators of the rumen microbiome

“…Nevertheless, the results are mixed, and while ruminal ciliate contributions to nitrate reduction have been noted during methane mitigation, an understanding of the mechanisms involved remains incomplete. Further, an enhanced metabolic reduction in nitrate and nitrite has been observed in faunated conditions in both in vitro and in vivo studies [ 62 ], with ciliates and associated bacteria potentially contributing to nitrite reduction [ 63 ]. This interaction highlights the role of ruminal ciliates in ensuring the safe application of nitrate for methane mitigation.…”