Toward Standardisation of Methodologies and Biodiversity Measures for Describing the Microbiome Structure and Diversity of Silages

Abstract

Ensiling is a widely adopted fermentation method for forage preservation that relies on the dynamic succession of microorganisms; ultimately, lactic acid bacteria (LAB) dominate a successful fermentation. This review conducts a meta-analysis of studies published from 2019 to 2024 and identifies critical gaps in methodological standardisation. The majority of studies focus on traditional temperate forages, while nonconventional tropical forages and agro-industrial residues remain underexplored. In light of climate change and rising temperatures, understanding the impacts of high-temperature ensiling on microbial communities is crucial, as these conditions may increase the alpha diversity of the resulting silage and enhance heterofermentative fermentation. Comparison between studies is impeded by variations in silage technology, DNA extraction protocols, and bioinformatic pipelines. We propose the development of MOCK communities to be shared among laboratories for their inclusion in silage DNA extractions, for rigorous assessments of DNA lysis efficiency, and a shift toward high-resolution clustering approaches (e.g., amplicon sequence variants and, when appropriate, a 99% cutoff). Moreover, the use of Hill numbers is encouraged as a robust alternative for expressing alpha diversity. This review not only synthesises current best practices for microbiome analysis in silages but also provides recommendations for reproducibility and consistency in future research, thereby aiding decision-making to optimise fermentation outcomes.