Exploring the fecal microbiota of Ragusano donkey

The “Ragusano” is a donkey breed reared on the Mediterranean island of Sicily (Italy), where for a long time it has played an important cultural role both as a working animal, for carrying goods, and for its high-quality meat and milk which are still consumed by people today 1,2]. Recently, thanks to the development of the high-throughput DNAs sequencing techniques, most of the scientific researches focused their attention in understanding the taxonomic diversity of microbal communities populating animal gastrointestinal tracts, as the gut microbiota is known to play a crucial role in modulating host health [3,4,5,6]. The intestinal tract of animal belonging to the Equidae family is colonized by a diverse community of microorganisms such as bacteria, archea, fungi, parasites, protozoa, and viruses. Unfortunately, no data are currently available about microbial diversity of ragusano donkey gut microbiota. For this reason, the aim of this study was that to characterize, for the first time, the fecal microbiota of this autochthonous donkey, in order to explore the taxonomic and functional profiles of gut microbes potentially associated with this animal. Genomic DNA was extracted from each fecal sample taken directly from the rectal ampoule of 18 female of Ragusano donkeys, officially registered in “Herd Book”. The V3-V4 region of the bacterial 16S rDNA gene and the ITS1 fungal region using paired-end strategy (2 x 300 bp) on an Illumina Miseq platform. Bioinformatics analysis included raw reads processing “Amplicon Sequence Variants" (ASVs) identification and classification, functional prediction and statistical analysis. In this metagenomic study, a total of 23 phyla, 190 families and 350 bacterial genera were identified using an identity threshold of 74%, 85% and 95% respectively; of these, only 11 phyla, 69 families and 125 genera showed a relative abundance greater than 0.1%. The most abundant phylum was Bacteroidetes (~44%) followed by Firmicutes (~40.8%), Spirochaetes (~8.9%), Fibrobacteres (~3.2%) and Proteobacteria (~2%). However, this trend was not always confirmed as in some fecal samples (6/18 samples), the most abundant phylum was that of Firmicutes, with an abundance rate ranging from ~42% to ~51%. Regarding fungal diversity, our analysis identified a total of 11 phyla, 125 families and 165 fungal genera of which 9 phyla, 59 families and 70 genera showed a relative abundance rate higher than 0.01%. At the phylum level, Basidiomycota was the most abundant fungal group (~36.6%) followed by Neocallimastigomycota (~24%), Ascomycota (~11.3%), and Mucoromycota (~3.7). Overall, the most abundant phylum/microbial families (both bacteria and fungi) found in this study reflect the remarkable ability of donkeys to better digest fibers. The complex function of Ragusano donkey’ s microbiota is further supported by prediction of thousands of enzymatic classes involved in a number of biochemical and molecular pathways. In fact, in this study over 2000 enzymatic classes and 404 metabolic pathways were identified. Overall, the results of this study provide a first overview of the microbial communities colonizing the intestinal tract of the Ragusano donkey. Studies like this, offer a cutting-edge tool for the development of optimal animal husbandry and management solutions, as well as useful information for the welfare and enhancement of the Ragusano donkey breed. The obtained results are encouraging and may serve as a starting point for future microbiome investigations in this endangered autochthonous Sicilian donkey breed.