Lactoferrin modulates hydrogen flow and methane output during in vitro rumen fermentation

Lactoferrin (LF) is a naturally milk-derived glycoprotein with antimicrobial and immunomodulatory properties; however, its roles in rumen fermentation and methane production remain unclear. This study evaluated the reproducibility and temporal dynamics of bovine LF supplementation using independent in vitro rumen fermentation experiments. Six batch culture experiments were conducted with rumen fluid collected from different donor cows and diets to capture biological variability. Fermentations were performed in sealed serum bottles under anaerobic conditions, with LF supplemented at graded doses of 2.5–7.5 g per bottle (1.2 g substrate in 150 mL incubation volume). Gas production was monitored, and methane (CH 4 ) and carbon dioxide (CO 2 ) were quantified by gas chromatography at 24 and 48 h. LF consistently reduced the proportion of methane in total gas (p < 0.05), with significant effects at 24 h, while absolute methane production decreased significantly at 24 h for 2.5–5.0 g doses (greatest ∼25%; p < 0.05), but reductions at 48 h were modest and non significant. LF increased total gas production and shifted fermentation toward higher CO 2 , propionate, and butyrate formation (p < 0.05), accompanied by reduced acetate-to-propionate ratios, while rumen pH and dry matter degradation were largely maintained. Microbial community composition and methanogenesis-associated functional potential were assessed using full-length 16S rRNA gene sequencing coupled with FAPROTAX-based functional prediction. An LF dose of 5.0 g revealed distinct compositional shifts and reduced methanogenesis-associated functional potential. Collectively, these findings demonstrate that LF exerts reproducible and time-dependent effects on rumen fermentation and methane proportion in vitro, supporting its further in vivo evaluation.