Production of N2 and N2O from nitrate ingested by sheep
An earlier in vitro study using 13N and 15N isotopes showed that a small fraction of supplemental JOURNAL/japan/04.02/00032417-201801000-00058/math_58MM8/v/2018-02-02T025546Z/r/image-png was converted to N2O, but concluded that denitrification to N2 did not occur (Kaspar & Tiedje, 1981). More recently, however, a significant number of genes coding for denitrifying enzymes have been identified in the bovine rumen (Brulc et al., 2009), suggesting that denitrification might be more significant than previously thought (Latham, Anderson, Pinchak, & Nisbet, 2016). We observed in vitro that N2O accumulated only in rumen fluid withdrawn from sheep that were not adapted to dietary JOURNAL/japan/04.02/00032417-201801000-00058/math_58MM9/v/2018-02-02T025546Z/r/image-png supplementation; in addition, N2O produced disappeared from the headspace gas towards the end of the incubation period suggesting denitrification was occurring (de Raphélis‐Soissan, Nolan, Newbold, Godwin, & Hegarty, 2016). We hypothesised that the occurrence of N2O emission depends on ruminal adaptation to JOURNAL/japan/04.02/00032417-201801000-00058/math_58MM10/v/2018-02-02T025546Z/r/image-png and that N2O may be further converted to N2. To test this hypothesis, we administered 15JOURNAL/japan/04.02/00032417-201801000-00058/math_58MM11/v/2018-02-02T025546Z/r/image-png intraruminally to sheep that were either adapted to dietary JOURNAL/japan/04.02/00032417-201801000-00058/math_58MM12/v/2018-02-02T025546Z/r/image-png or non‐adapted to JOURNAL/japan/04.02/00032417-201801000-00058/math_58MM13/v/2018-02-02T025546Z/r/image-png but instead were given the same basal diet with an iso‐nitrogenous urea supplement. After dosing, sheep were moved immediately into gas‐tight portable accumulation chambers (PACs; Goopy, Woodgate, Donaldson, Robinson, & Hegarty, 2011) that enabled emissions of N2O, CO2 and CH4 to be monitored and 15N enrichments in N2, and N2O to be determined.