TITLE:
The Effect of Sources of Rumen Fluid and Dissolved Oxygen Concentration on Rumen Fermentation Parameters, Methane Gas Emissions, and Microbiota Diversity Changes in Laboratory Conditions
AUTHORS:
Byeng Ryel Min, Mariline Hilaire, Santosh Chaudhary, Heba Abdo, Hossam Ismael, Ryszard Puchala
KEYWORDS:
Bacteroidetes, Firmicutes, Methanogens, Methane, Oxygen
JOURNAL NAME:
Open Journal of Animal Sciences,
Vol.16 No.4,
October
19,
2026
ABSTRACT: This study assessed the impact of rumen fluid source, basal diet, and dissolved oxygen content in rumen liquor on in vitro rumen fermentation, ruminal gas production, and changes in ruminal microbiota diversity. Two sets of in vitro experiments were conducted. The first study examined the effects of sources of individual or equally pooled rumen liquors (Exp. 1) on ruminal pH, greenhouse gas (GHG) emissions, and microbiota changes. The second part of the experiment (Exp. 2) focused on assessing how different levels of dissolved oxygen (O2) and different diets (grain-based versus forage-based) influence in vitro digestibility (IVDMD), methane (CH4) and other gas emissions, rumen fermentation, and microbial populations. All gases were collected using an ANKOM Gas Production System to analyze CH4, nitrous oxide (N2O), and hydrogen sulfide (H2S). Triplicate qPCR analyses were used to evaluate microbial diversity in the rumen of cannulated steers. Incubations with mixed rumen fluid (RF) produced more gas (P 4 and H2S production, Firmicutes (F), Bacteroidetes (B), F/B ratio, and total methanogens were at intermediate levels with mixed RF compared to individual donor steer samples. The dissolved O2 treatment did not affect the average in vitro dry matter disappearance rate (IVDMD), with no significant diet × treatment interactions. Increasing O2 supply reduced (P 4 (mL/24h), and H2S (mL/24h) production in the forage-based diet, with no significant diet × treatment interaction (P > 0.05). The findings indicated that RF obtained from a single animal was prone to error due to host-animal variation and suggested that mixed RF from multiple donor animals can be used in an in vitro study. Dissolved O2 levels in the rumen and diets significantly influenced rumen gas production, resulting in different fermentation end products, microbiome diversity, and total methanogen abundance.