PSXI-30 Ruminal Bacterial Diversity Indexes of Grazing Beef Cattle Receiving Different Supplements Plus Phytogenic Additives
Authors: Camargo Karine Dalla Vecchia , Siniscalchi Débora , Fonseca Natalia Vilas Boas , De Souza Willian Aparecido , Castro Marcelo Augusto Oliveira , Afonso Barbara Carolina , Dornellas Isadora Alves , Umaña Fiorella Viquez , Prados Laura Franco , Paulino Pedro Veiga Rodrigues , Granja-Salcedo Yury Tatiana , Mantovani Hilário Cuquetto , Reis Ricardo 
Published: November 2023

Abstract
The addition of phytogenic compounds that have antimicrobial activity in cattle supplements can promote changes in ruminal bacterial diversity. This study aimed to evaluate the effect of two levels of supplementation plus a blend of phytogenic additives (essential oils, saponins, and spices) on the measures of alpha and beta diversity in the rumen of beef cattle grazing tropical pasture. The experiment was carried out in the Beef Cattle Sector of UNESP, Jaboticabal-BR. The trial lasted 84 days and nine castrated Nellore steers (body weight of 262 ± 31.2 kg) cannulated in the rumen were assigned in 3 simultaneous 3 × 3 Latin square designs. Animals were kept on the pasture of Urochloa brizantha cv. Marandu in the rainy season. The treatments were: ad libitum mineral supplementation (MS) as control group; added mineral supplementation, 0.1% of body weight per day plus Cargill phytogenic blend (AMSP); and energetic-protein supplementation, 0.3% of body weight per day plus Cargill phytogenic blend (EPSP). Ruminal samples were collected on day 26 of each experimental period. A mix of liquid and solid rumen content (6 mL) was taken before supplementation, placed in cryogenic tubes, and immediately frozen in liquid nitrogen. All samples were stored at −80 °C until DNA extraction and analysis. Total genomic DNA was extracted using the Quick-DNA Fecal/Soil Microbe Miniprep kit. The V3-V4 hypervariable regions of the bacterial 16S rRNA gene were amplified primers 341F (CCTACGGGNGGCWGCAG) and 806R (GGACTACHVGGGTWTCTAAT). The PCR fragments were submitted to sequencing on an Illumina NovaSeq 6000 PE 250 platform. The sequences were processed using mothur, grouped into operational taxonomic units (OTUs) using a similarity cut-off value of 97%, and the bacterial OTUs were classified using the SILVA database. Alpha diversity indices including Shannon, Simpson, Fisher, Chao1, and ACE, were calculated using the phyloseq package, while beta diversity was determined using the vegan package, both in Rstudio. Alpha diversity indices were compared between treatments using a non-parametric Kruskal–Wallis test and Wilcoxon’s post-hoc test. Beta diversity was tested using permutational multivariate analysis of variance (PERMANOVA) based on the Bray-Curtis similarity index. The AMSP and EPSP did not affect measures of alpha diversity (Figure 1), such as Shannon and Fisher indices (P = 0.219) as well as the richness index measured using Chao1 and ACE (P ≥ 0.363). A negative effect of supplementation on alpha diversity (P = 0.004) was only observed with Simpson’s Index of Diversity. Additionally, the supplementation did not change the beta-diversity of the ruminal bacterial community based on Bray–Curtis similarity index (P = 0.510; Figure 2). In conclusion, the addition of phytogenic compounds in the two levels of supplementation showed only a minor impact on the alpha diversity in the rumen of grazing beef cattle.