Isotopic H2 and NH4+ supplementation enhances volatile fatty acid and microbial protein synthesis during in vitro rumen fermentation

The volatile fatty acid (VFA) and microbial protein production are tightly linked to hydrogen (H 2 ) metabolism in rumen. The H 2 can be split by hydrogenases into protons and electrons, and is regarded as the most primordial source of reducing equivalents within the rumen microecosystem. However, the quantitative role of H 2 in VFA and microbial protein formation has not been thoroughly studied. Additionally, while rumen fermentation is influenced by ammonia-N availability, the combined effects of H 2 and NH 4 + supplementation on VFA and microbial protein synthesis have not been fully explored. The objective of this study was to explore the role of proton and electron transfer from H 2 in VFA and microbial protein synthesis based on the synergistic effect of H 2 and NH 4 + supplementation. In vitro ruminal batch culture was employed for the complete block design with factorial arrangement of four treatments including H 2 (0 or 40 mL 2 H 2 ) and NH 4 + (0 or 8 mmol/L 15 NH 4 + ) supplementations. 2 H 2 supplementation increased dry matter (DM) degradation ( P = 0.005), methane (CH 4 ) production ( P < 0.001), 2 H abundance ( P < 0.001) in microbial protein and individual VFAs. However, 2 H abundance in microbial protein, acetate, propionate and butyrate are low with the value less than 2.0‰. The 15 NH 4 + supplementation increased microbial protein production ( P = 0.001), microbial 15 N abundance ( P < 0.001), and microbial 15 N incorporation (mg/g degraded DM, P < 0.001), and concurrently reduced CH 4 production ( P < 0.001). Moreover, 2 H 2 supplementation further enhanced the stimulatory effect of 15 NH 4 + on microbial protein production. These results indicate limited incorporation of protons for the synthesis of VFA and microbial protein, and the enhancement of VFA and microbial protein production may be associated with increased electron availability caused by the 2 H 2 supplementation. The isotopic dilution and hydrogen exchange with other pools cannot be ruled out as contributing factors, which should be explored in the future studies.