This study aimed to investigate the effects of two practically relevant doses of menthol-rich plant bioactive lipid compounds (PBLC) on fermentation, microbial community composition, and their interactions in sheep rumen. Twenty-four growing Suffolk sheep were divided into three treatments and were fed hay ad libitum plus 600 g/d of concentrate containing no PBLC (Control) or PBLC at low dose (80 mg/d; PBLC-L) or high dose (160 mg/d; PBLC-H). After 4 weeks on the diets, samples of ruminal digesta were collected and analyzed for short-chain fatty acid (SCFA), ammonia, and microbiota; microbiota being analyzed in the solid and the liquid digesta fractions separately. Ruminal SCFA and ammonia concentrations were not affected by the PBLC treatments. The microbiota in the solid fraction was more diverse than that in the liquid fraction, and the relative abundance of most taxa differed between these two fractions. In the solid fraction, phylogenetic diversity increased linearly with increased PBLC doses, whereas evenness (lowest in PBLC-L) and Simpson diversity index (greatest in PBLC-H) changed quadratically. In the liquid fraction, however, the PBLC supplementation did not affect any of the microbial diversity measurements. Among phyla, Chloroflexi (highest in PBLC-L) and unclassified_bacteria (lowest in PBLC-L) were altered quadratically by PBLC. Lachnospiraceae, Bacteroidaceae (increased linearly), BS11 (increased in PBLC-L), Christensenellaceae (decreased in PBLC treatments), and Porphyromonadaceae (increased in PBLC treatments) were affected at the family level. Among genera, Butyrivibrio increased linearly in the solid fraction, YRC22 increased linearly in the liquid fraction, whereas Paludibacter increased and BF311 increased linearly with increasing doses of PBLC in both fractions. The PBLC treatments also lowered methanogens within the classes Thermoplasmata and Euryarchaeota. Correlation network analysis revealed positive and negative correlations among many microbial taxa. Differential network analysis showed that PBLC supplementation changed the correlation between some microbial taxa and SCFA. The majority of the predicted functional features were different between the solid and the liquid digesta fractions, whereas the PBLC treatments altered few of the predicted functional gene categories. Overall, dietary PBLC treatments had little influence on the ruminal fermentation and microbiota but affected the associations among some microbial taxa and SCFA
