Chitosan reduces methane emissions and alters the phylogenetic affiliation of sampled methanogens in in vitro rumen...

Chitosan has been extensively investigated as a natural feed additive in ruminant diets; however, its effects on ruminal fermentation and enteric methane (CH₄) mitigation remain inconsistent. This study aimed to evaluate the impacts of graded chitosan supplementation on in vitro digestibility, gas and CH₄ production, rumen fermentation characteristics, protozoal populations, and the phylogenetic affiliation of the recovered methanogens. A total mixed ration (50% forage:50% concentrate) was incubated for 24 h with three chitosan doses-CHI4, CHI8, and CHI12 (4, 8, and 12 g/kg DM, respectively)-and a control. Gas production kinetics were assessed using a semi-automated in vitro gas production system, and methanogen-specific 16S rRNA genes were amplified by nested PCR and sequenced using Sanger sequencing to generate individual consensus sequences for phylogenetic analysis rather than operational taxonomic units (OTUs) derived from high-throughput sequencing. Chitosan at 8 and 12 g/kg DM linearly reduced net gas production (P < 0.001) and CH₄ output (P = 0.008), with CHI8 yielding the greatest reduction (11%) in CH₄ compared to the control. True dry matter degradation (TDDM; P = 0.032) and true organic matter degradation (TDOM; P = 0.012) were significantly decreased by CHI8 and CHI12. Partitioning factor (PF) values were increased with CHI8 supplementation (P < 0.001 linear; P = 0.021 quadratic). All chitosan treatments elevated ammonia-N concentrations (P < 0.001). CHI12 significantly increased total volatile fatty acids (VFAs; P = 0.001), acetate (P = 0.016), propionate (P = 0.016), valerate (P = 0.001), and branched-chain VFAs (P < 0.01), whereas CHI8 reduced butyrate levels (P = 0.018), and selectively modulated protozoal populations (with reductions in Epidinium spp. and Eudiplodinium spp.). Phylogenetic analysis of methanogen 16S rRNA sequences revealed treatment-associated differences in the affiliation of the limited number of sequences recovered, with CHI4 and the control grouped with Uncultured Methanobacteriaceae, while CHI8 and CHI12 aligned with Uncultured Methanobrevibacter strains. Comparative analysis showed high sequence similarity between chitosan-derived isolates and reference sequences from North America. In conclusion, chitosan supplementation induced qualitative shifts in the phylogenetic affiliation of the sampled methanogens, with supplementation at 8 g/kg DM effectively reduced CH₄ emissions, whereas 12 g/kg DM provided additional improvements in ruminal fermentation characteristics.