Oregano Essential Oil Alleviates Diquat-Induced Oxidative Stress in Calves and Modulates Rumen Microbiota

Abstract

Oxidative stress can impair calf health and performance, and oregano essential oil (OEO) has potential antioxidant activity. This study evaluated dietary OEO in a diquat-induced oxidative-stress model using 24 weaned male Holstein calves assigned to a 2 × 2 factorial design with OEO supplementation (0 or 8 g/day) and diquat challenge (saline or 8 mg/kg body weight). Growth performance, serum biochemical, antioxidant, oxidative-damage and inflammatory indices, hepatic Nrf2-related antioxidant gene expression, rumen microbiota, and rumen metabolomics were assessed. The results showed that growth responses were phase dependent: OEO increased overall average daily gain (ADG) and dry matter intake (DMI), whereas diquat reduced overall ADG; no OEO × diquat interaction was detected for overall growth outcomes. The OEO group had higher serum SOD, GSH-Px, and T-AOC levels and lower levels of selected oxidative-damage and inflammatory indices, although the responses varied by endpoint and time point. Hepatic RT-qPCR showed treatment-associated differences in Keap1, HMOX1, GCLC, GCLM, and SOD1 expression. Rumen 16S rRNA gene sequencing showed treatment-associated shifts in microbial community composition, whereas PICRUSt2 predicted differences in microbial functional potential, including amino-acid-metabolism-related functions. Untargeted metabolomics identified treatment-associated differences in rumen metabolite profiles and KEGG pathway-enrichment patterns. Correlation analysis indicated that OEO-associated characteristic metabolites were positively correlated with antioxidant markers and negatively correlated with oxidative-damage and pro-inflammatory indices. Overall, OEO supplementation was associated with improved antioxidant status and distinct rumen microbial-metabolic profiles in diquat-challenged calves, supporting its potential as a natural feed additive, while the involvement of specific molecular pathways requires further validation.