The Impact of Mineral and Energy Supplementation and Phytogenic Compounds on Rumen Microbial Diversity and Nitrogen Utilization in Grazing Beef Cattle

**Background:** Rumen microbiota activity is crucial for nutrient supply in cattle. Antimicrobial feed additives have been banned due to resistance concerns, prompting interest in phytogenic alternatives like tannins and essential oils. Hydrolyzable tannins at low-to-moderate levels (≤20 g/kg DM) may reduce nitrogen losses and improve VFA profiles. Essential oil compounds such as cinnamaldehyde and carvacrol have antimicrobial properties that could modulate rumen fermentation. However, the combined effects of these phytogenic compounds in energy-supplemented grazing beef cattle on rumen microbiota and nitrogen metabolism had not been reported. This study hypothesized that a blend of hydrolyzable tannins, carvacrol, and cinnamaldehyde oil in energy supplements would improve nitrogen metabolism by modulating the rumen microbiome and reducing urinary nitrogen excretion.

**Methods:** Eight castrated Nellore steers (456.6 ± 32.8 kg) with rumen cannula were randomly assigned in a double 4×4 Latin square with a 2×2 factorial arrangement (energy vs. mineral supplement; with vs. without phytogenic compounds). Four 28-day periods (14 d adaptation, 14 d sampling) were conducted during the rainy season (January–April 2018) in Brazil. Steers grazed Urochloa brizantha cv. Marandu under continuous stocking. Energy supplements (EW and EPHA) were offered at 0.3% BW daily; mineral supplements (MW and MPHA) were ad libitum. The PHA blend contained 10% carvacrol and cinnamaldehyde oil and 90% hydrolyzable tannins from berries and grapes, supplied at 1.5 g/kg ingested DM. Forage intake and digestibility were estimated using chromium oxide (external marker) and iNDF (internal marker). Urine spot samples were collected for nitrogen metabolite analysis (creatinine, uric acid, urea, allantoin, total N). Rumen fluid was collected at 0, 6, 9, and 12 h post-supplementation for pH, NH3-N, and VFA analysis. Rumen content for microbiota analysis was collected pre-supplementation; DNA was extracted and the V4–V5 region of 16S rRNA was sequenced on Illumina MiSeq. Bioinformatics used QIIME 2 with DADA2 denoising and Silva 128 taxonomy assignment.

**Key Results:** Energy supplementation significantly increased total DM intake (2.52% vs. 1.87% BW; p = 0.001), OM intake (p = 0.003), CP intake (p = 0.006), apNDF intake (p = 0.017), and gross energy intake (p = 0.004). Apparent total digestibility of DM (67.0% vs. 62.5%; p < 0.001), OM (71.0% vs. 64.0%; p < 0.001), CP (70.0% vs. 58.9%; p < 0.001), apNDF (72.5% vs. 66.2%; p < 0.001), and GE (68.5% vs. 60.0%; p < 0.001) were all higher with energy vs. mineral supplements. There was a tendency for lower DM digestibility with PHA addition (64.8% vs. 62.8%; p = 0.073). A significant interaction (p = 0.033) showed that steers on EW had higher urinary N excretion (116.3 g/d) vs. MW (89.7 g/d) and MPHA (96.1 g/d). Energy supplements increased N intake (p = 0.006), N retained (g/d and % of N intake; p < 0.001 and p = 0.050), and plasma urea N (p < 0.001). Ruminal NH3-N was higher with energy supplements (16.51 vs. 14.23 mg/dL; p = 0.003). PHA addition reduced ruminal valerate proportion (1.06% vs. 1.15%; p = 0.015) and tended to increase propionate (16.80% vs. 16.69%; p = 0.074). Beta-diversity (Bray–Curtis) differed between energy and mineral supplement groups (p = 0.015). Energy supplements increased Proteobacteria (p = 0.046) and Spirochaetae (p = 0.022) phyla, with tendencies for higher Bacteroidetes (p = 0.089) and lower Firmicutes (p = 0.069). PHA reduced Verrucomicrobia (p = 0.022). At the family level, PHA increased Bacteroidales BS11 gut group (p = 0.024) and reduced Veillonellaceae in EPHA vs. other groups (p = 0.007). Several genera were affected by interactions, including lower Roseburia and Family XIII UCG-002 with MPHA. PHA reduced Ruminiclostridium 6 (p = 0.011).

**Clinical Implications:** Energy supplementation at 0.3% BW is an effective strategy to improve intake, digestibility, and nitrogen retention in beef steers grazing tropical forages during the rainy season. However, the addition of this specific phytogenic blend (hydrolyzable tannins, carvacrol, cinnamaldehyde) at 1.5 g/kg DM did not improve nitrogen utilization or microbial protein synthesis efficiency and negatively impacted total DM digestibility and the abundance of several cellulolytic bacterial groups. The findings suggest that this dose and combination of phytogenic compounds is not beneficial for grazing beef cattle under these conditions. Further research is needed to explore different doses and blends of phytogenic compounds to determine if beneficial effects on rumen fermentation and nitrogen efficiency can be achieved without compromising digestibility.