Can adaptation to nitrate supplementation and provision of fermentable energy reduce nitrite accumulation in rumen contents in vitro?

Abstract

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.

Production of N2 and N2O from nitrate ingested by sheep

“…The regulatory mechanisms of denitrification pathways in the rumen are not yet well understood. Our recent in vitro study suggested that N 2 O only accumulates in incubation media when rumen fluid supplemented with was taken from donors that were not adapted to dietary (de Raphélis‐Soissan et al., ). In the current study, however, there was no difference in 15 N partitioning between adapted and non‐adapted sheep.…”

“…Both and concentrations were determined in formaldehyde preserved samples by a continuous‐flow analyser (San++, Skalar, Breda, the Netherlands) as described by de Raphélis‐Soissan et al. ().…”

“…More recently, however, a significant number of genes coding for denitrifying enzymes have been identified in the bovine rumen (Brulc et al., ), suggesting that denitrification might be more significant than previously thought (Latham, Anderson, Pinchak, & Nisbet, ). We observed in vitro that N 2 O accumulated only in rumen fluid withdrawn from sheep that were not adapted to dietary supplementation; in addition, N 2 O produced disappeared from the headspace gas towards the end of the incubation period suggesting denitrification was occurring (de Raphélis‐Soissan, Nolan, Newbold, Godwin, & Hegarty, ). We hypothesised that the occurrence of N 2 O emission depends on ruminal adaptation to and that N 2 O may be further converted to N 2 .…”

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

“…Furthermore, addition also stimulated accumulation at a greater rate in the SOL fraction of DEF digesta relative to both fractions of FAU digesta suggesting that incoming slows reduction to NH 3 in the absence of protozoa (Lin et al, 2011; Yoshida et al, 1982). The accumulation of NH 3 in WRF after addition of relative to CON may be explained by the fact that both and reductase activity increases in response to exposure in rumen digesta from adapted sheep (Alaboudi & Jones, 1985; Shi et al, 2012) although this effect has not always been reported in the literature (de Raphélis‐Soissan, Nolan, Newbold, Godwin, & Hegarty, 2016a).…”

Low Emission Feed : using feed additives to decrease methane production in dairy cows

“…Aschenbach et al (2009) reported that nitrate impaired acetate uptake through the rumen wall in vitro. Nolan et al (2016) suggested this to be a possible explanation for a shift in VFA profile towards acetate that has been reported upon nitrate supplementation, both in vitro and in vivo (Zhou et al, 2012;Guyader et al, , 2016de Raphélis-Soissan et al, 2016a). However, in the study of Aschenbach et al (2009) only acetate uptake was measured, and not uptake of propionate and butyrate.…”