To study bacterial shifts during the bloat process, 2 dietary interventions for alleviating the severity of bloat on rumen bacterial composition were studied in 12 rumen-cannulated cattle in a 3-phase crossover study. Cattle were subjected to 1 of 3 treatments: (1) pure alfalfa pasture (PA), (2) pure alfalfa pasture supplemented with Alfasure (AA), and (3) alfalfa/sainfoin mixed pasture (AS). A 7-d washout interval was provided between phases during which cattle received a baseline diet. Rumen liquid and solid fractions were collected from non-bloated (NB; bloat score = 0) and bloated (B; bloat scores = 1–3) cattle within each treatment group and subjected to genomic DNA extraction and Illumina sequencing of the V3-V4 regions of bacterial 16S rRNA gene. On average, 47,211 high-quality sequences were generated per sample. Taxonomic classification revealed the presence of 20 different bacterial phyla among which Bacteroidetes followed by Firmicutes, Fibrobacteres, Cyanobacteria, and Spirochaetes were identified as predominant (>1% of population) members of rumen microbiota. The PERMANOVA analysis of UniFrac distances revealed distinct (P < 0.05) clustering patterns when comparing PA(B), AA(NB) and AS(NB)-associated microbiota. Statistical analysis (LEfSe) of phylogenetic data for rumen liquid and solid fractions also revealed significant associations between several members of microbiota and treatment groups (P < 0.05; Table 1). Further investigation can focus on manipulation of rumen microbiota in favor of increased bloat-resistant functional properties. Table 1. Association of bacterial genera with treatment groups¹

Genus		Phylum		Treatment group2
Rumen liquid fraction
Succinivibrio		Proteobacteria		PA(B)
Succiniclasticum		Firmicutes		PA(B)
Butyrivibrio		Firmicutes		AS(NB)
Coprococcus		Firmicutes		AS(NB)
Fibrobacter		Fibrobacteres		AA(NB)
Ruminococcus		Firmicutes		AA(NB)
Rumen solid fraction
Butyrivibrio		Firmicutes		PA(B)
Streptococcus		Firmicutes		PA(B)
Prevotella		Bacteroidetes		AS(NB)
Fibrobacter		Fibrobacteres		AA(NB)

¹Linear discriminant analysis effect size (LEfSe) was used to test for significant associations (P < 0.05, and >3 log fold increase in the relative abundance). ²PA(B) = pure alfalfa (bloated); AS(NB) = alfalfa-sainfoin (non-bloated); AA(NB) = alfalfa-Alfasure (non-bloated).