Simple SummaryGlobally, the livestock sector is responsible for 37% of total anthropogenic methane emissions, most of which are produced from enteric fermentation of ruminants. Livestock is also responsible for 65% anthropogenic nitrous oxide and 64% of anthropogenic ammonia emissions. The literature reports several dietary management options to reduce greenhouse gas emissions from ruminants, and potentially improve productivity. However, strategies that aim to reduce the emissions of one specific greenhouse gas can have side effects (increase) on other pollutant gases. In this study, we evaluated the effect of two types of perennial ryegrass (PRG) pastures differing in their concentration of water-soluble carbohydrates (WSC, high (HS) and low (LS)) on the in vitro nitrogen use efficiency in the rumen and on methane emissions. The greater WSC and lower crude protein (CP) concentrations of high sugar pastures modified in vitro rumen fermentation, tending to increase total volatile fatty acids (VFA) production, reduce acetate:propionate ratio and methane (CH4) concentration, and improve nitrogen (N) use efficiency through lower rumen ammonia-N (NH3-N) concentrations. In vivo studies with cattle are required to confirm the potential of these measures to increase the sustainability and reduce the environmental impact of grazing livestock production systems.The objective of this study was to determine the effect of perennial ryegrass (PRG) forages differing in their concentration of water-soluble carbohydrates (WSC) and crude protein (CP), and collected in spring and autumn, on in vitro rumen fermentation variables, nitrogen (N) metabolism indicators and methane (CH4) output, using a batch culture system. Two contrasting PRG pastures, sampled both in autumn and spring, were used: high (HS) and low (LS) sugar pastures with WSC concentrations of 322 and 343 g/kg for HS (autumn and spring), and 224 and 293 g/kg for LS in autumn and spring, respectively. Duplicates were incubated for 24 h with rumen inocula in three different days (blocks). Headspace gas pressure was measured at 2, 3, 4, 5, 6, 8, 10, 12, 18, and 24 h, and CH4 concentration was determined. The supernatants were analysed for individual volatile fatty acids (VFA) concentrations, and NH3-N. The solid residue was analysed for total N and neutral detergent insoluble N. Another set of duplicates was incubated for 4 h for VFA and NH3-N determination. The HS produced more gas (218 vs. 204 mL/g OM), tended to increase total VFA production (52.0 mM vs. 49.5 mM at 24 h), reduced the acetate:propionate ratio (2.52 vs. 3.20 at 4 h and 2.85 vs. 3.19 at 24 h) and CH4 production relative to total gas production (15.6 vs. 16.8 mL/100 mL) and, improved N use efficiency (22.1 vs. 20.9). The contrasting chemical composition modified in vitro rumen fermentation tending to increase total VFA production, reduce the acetate:propionate ratio and CH4 concentration, and improve N use efficiency through lower rumen NH3-N.