Cows relocation affects exosomes and their cargos in bovine milk

Extracellular vesicles in milk contains several compounds, as lipids, proteins, noncoding RNAs, and mRNAs. Among the different types of extracellular vesicles, exosomes have attracted researchers since their sorting cargos is a regulated, nonrandom process, which is heavily involved in cell-to-cell communication. The aim of the study was to analyse the variations of RNAs in milk exosomes in response to the relocation of dairy cows between production groups. The study was carried out on 76 lactating cows during the relocation from high yielding group to low yielding group. The extent of stress was evaluated by means of milk cortisol in samples collected in the afternoon from 2 days before to 3 days after the change of the group. Mean cortisol concentrations significantly increase on the day of relocation (day 3) and on the days 4 and 5. A subgroup of 6 cows were selected according to cortisol variations after the relocation. Cows were ranked according to cortisol increase after relocation and the 3 cows with the highest variation and the 3 cows with the lowest variation were selected. Pools of Milk samples of days 1 and 2 (Before) and days 4 and 5 (After) were prepared for exosomes isolation using a commercial kit (ExoEasy Maxi Kit, QIAGEN). Exosomes were visualized with transmission electron microscopy (TEM) after immunolabeling followed by negative stain and electron microscopy was carried out targeting CD63 and HSC70 and analyzed on Philips CM10. Images were recorded at 80 kV. Total extracted RNAs were quality checked using fluorometric quantification Qubit and processed with Illumina® TruSeq® Small RNA Library Prep protocol. Libraries were sequenced to 50 bp using an Illumina HiScan SQ. The mean number of sequences for the 12 samples was 64 millions of reads. Sequences were mapped on GCA_000003205.6_Btau_5.0.1 with Rsubread, annotated with GCF_000003205.7_Btau_5.0.1_genomic.gff and compared using Bioconductor DESeq2 software. Overall, 9 miRNA and 7 lncRNA resulted differentially (P<0.05 FDR) expressed between Before and After groups, suggesting a potential role of exosome cargos as a proxy for animal resilience