PSXIII-20 - The effect of hemp varieties and cannabidiol levels on the in vitro ruminal microbiome

Abstract: This study aimed to evaluate the effect of hemp varieties and cannabidiol (CBD) levels on in vitro ruminal microbiota diversity and abundance relative do alfalfa (Medicago sativa). Two ruminally cannulated Angus steers were used as inoculum donors for in vitro incubation. Two 160-mL serum bottles containing 200 mg air-dried of either alfalfa, fedora 17 (F17) and felina 32 after late harvest (F32S) or without seeds (F32O) were incubated in 14 mL of buffering media and 4 mL of rumen inoculum. A stock solution of 1mg/mL of CBD was prepared and added in increasing concentrations into each bottle to reach a final concentration of 0.001%, 0.002%, 0.003%, and 0.004% of CBD. After a period of 48 hours, the whole content inside of the serum bottles were used immediately utilized for DNA extraction followed by 16S rRNA sequencing with a Oxford Nanopore technologies MiniON. Amplicon sequence data were bioinformatically processed in QIIME2 version 2022.2. Alpha diversity was measured by the Shannon diversity index. Bray-Curtis’s dissimilarity matrix was constructed to estimate the differences among the samples and CBD levels, and differential abundance was then evaluated using analysis of compositions of microbiomes with bias correction (ANCOM) testing. Statistical significance was established at P < 0.05. All data were visualized in R version 4.1.2 (R Core Team, 2022) using the qiime2R package. Shannon diversity index was the highest for alfalfa (P < 0.05), but no difference was observed among the different CBD inclusion levels. When looking at beta diversity through Bray-Curtis, differences were only observed when comparing the different alfalfa and hemp varieties (P < 0.05), where alfalfa grouped separately from all the hemp samples. The most abundant phyla included Firmicute, Bacteroidetes, Proteobacteria, and Fusobacteria (P < 0.05). At family level, alfalfa samples had greater Aerococcaceae and Enterobacteriaceae than all the hemp treatments, but lower Lactobacillales (unclassified), Streptococcaceae, Succinivibrionaceae, and Bacilli (unclassified) than F32S and F17 (P < 0.05). In conclusion, these results suggest that sample type might play a bigger role on microbiome than CBD levels. Further studies are required to determine if higher levels of CBD than those examined herein could also lead to a shift on rumen microbes.