Effect of hydroponic barley fodder-containing diets on methane production and rumen fermentation in dairy cattle: an in vitro assessment

Water and arable land limitations, coupled with the urgent need to reduce enteric methane (CH4) emissions from the livestock sector, represent a major challenge to modern animal production systems. Hydroponic barley fodder (HBF) offers a resource-efficient, “landless” feed alternative, though its specific ruminal fermentation kinetics and methane mitigation potential remain largely uncharacterized. The objective of this study was to characterize the nutritional value and evaluate the enteric CH4 mitigation potential of HBF at two distinct harvest maturities when incorporated into cattle diets, validating its use in sustainable, land-independent ruminant production sys‑ tems. The HBF was collected on two different days of the growth cycle, on day 4 (T4: peak enzymatic activity and energy mobilization) and day 6 (T6: standard harvest maturity and maximum biomass) as critical metabolic inflection points. HBF was tested as a single ingredient (100% HBF) and in combination with hay and concen‑ trate at different inclusion levels (from 0 to 60%). In vitro fermentation was assessed utilising the Hohenheim Gas Test (HGT) technique. Individual rumen fluid from three fistulated cows was mixed with a buffer solution, and 200 mg of total substrate was incubated in glass syringes for 24 hours at 39 °C to measure pH, gas volume, gas composition, ammonia and digestibility. We hypothesized that HBF, being nutrient-dense and lower in fiber than standard hay, would favor propionate production (a hydrogen sink), thereby lowering methanogenesis. To definitively validate this metabolic shift, we evaluated the CH4/CO2 ratio (expressed as mL/L) and sampled the ruminal fluid to analyze Volatile Fatty Acid (VFA) profiles. While VFA analytical results are currently pending, the CH4/CO2 ratio serves as a crucial preliminary indicator: a lower ratio confirms a genuine shift towards hydro‑ gen-consuming pathways, proving that CH4 mitigation is achieved through improved energetic efficiency rather than merely reflecting a decrease in overall feed digestibility. Here, we show preliminary findings that align with our expectations. The pure substrates exhibited notably higher digestibility up to 71.6% and lower CH4/CO2 ratios (reduced by up to 6.8%) compared to the control diet (60% DM hay, 40% DM concentrate), furthermore, we observed a progressive dose-response effect in the hay-substitution diets. The observed difference in CH4 mit‑ igation potential between the harvesting stages suggests that the physiological maturity of T6 optimizes ruminal fermentation characteristics. Hence, our next step is to conduct final verification runs and integrate the pending VFA data to consolidate these promising gas kinetics and digestibility metrics. Preliminary in vitro data indicate that HBF is a highly digestible feed component with the potential to reduce the CH4 emissions. Therefore, replac‑ ing fibrous roughage with specific harvest maturities of hydroponic fodder is a promising strategy but needs to be verified in following in vivo studies