258 Investigating the Ruminal Metagenome of Neonatal Holstein Dairy Calves fed an Essential oil-Based Feed Additive
Authors: Fresno Rueda Anlly M , St-Pierre Benoit 
Published: October 2023

Abstract
Antibiotics remain the most effective tool for treating and preventing animal diseases in livestock production. However, the widespread use of antibiotics in food animals, as well as concerns about antibiotic resistance among bacterial pathogens, have raised awareness of the need to limit their use. As neonatal dairy calves are very susceptible to gastrointestinal infections that can result in high mortality and morbidity, the need to find alternatives to antibiotics has become a priority. Although essential oils (EO) have emerged as promising anti-microbial alternatives, their potential effects on the rumen microbiome are still poorly understood due to the complexity of their chemical components and limited understanding of their mechanisms of action. To gain further insight into the effects of EO on the bacterial communities of pre-weaned calves, we investigated the rumen metagenome of neonatal Holstein dairy calves that had been supplemented with 3.75g/feeding of a blend of EO (carvacrol, caryophyllene, p-cymene, cineole, terpinene, and thymol) as part of a previous study. Genomic DNA extracted from rumen contents collected from one of the EO-supplemented animals was used for high throughput sequencing using an Illumina Miseq (2x250) platform. Raw sequence reads were assembled into genomic contigs using custom-written Perl scripts. Coding sequences (CDS) were identified and annotated using RAST, with assignment of open reading frames (ORFs) to metabolic pathways using KEGG pathways. After assembly, a total of 3,052 contigs between 279 and 259,890 nt in length were obtained. From this set, 571 contigs affiliated to Prevotella ruminicola, representing 18.7% of total contigs built, were further analyzed. Data mining for specific functions within categories such as ‘protein metabolism’, ‘carbohydrates metabolism’, and ‘amino acids and derivatives’ was performed. This analysis resulted in the identification of all the enzymes involved in glycolysis. Two outcomes were predicted for pyruvate: conversion to lactate through lactate dehydrogenases (EC 1.1.1.27, EC 1.1.1.28), and the production of formate and acetyl-CoA through the activity of pyruvate formate-lyase (EC 2.3.1.54). Acetate (acetate kinase, EC 2.7.2.1) and propionate (Propionate kinase, EC 2.7.2.15) were other predicted end products. In addition to glucose fermentation pathways, enzymes potentially involved in the utilization of plant-based secondary metabolites were also identified, such as enzymes involved in Quinate-Chorismate metabolism: 3-dehydroquinate dehydratase (EC 4.2.1.10), Shikimate 5-dehydrogenase I alpha (EC 1.1.1.25), Shikimate kinase (EC 2.7.1.71), 5-Enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.19), and Chorismate synthase (EC 4.2.3.5). Identification of such metabolic pathways suggests that perhaps the supplemented EO additive could be used as a precursor for the synthesis of aromatic amino acids. Together, these findings indicate that EO supplementation may benefit animal health and performance by promoting the growth of bacterial species that can metabolize compounds in EO additives.