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Sheep faeces preservation affects microbiome activity

ABSTRACT: In vitro systems involving microbial fermentation such as the Hohenheim gas test (HGT) typically need freshly obtained inocula such as rumen fluid or faeces. The objective of this study was to test the possibility of becoming independent of fresh faeces by using preserved faeces. Fresh faeces from sheep (control, C) was compared with seven differently preserved faeces in the HGT by using nine feeds and studying in vitro gas production (GP). Preservation involved freezing at -20°C (FR), shock-freezing with liquid nitrogen (N) and additional freezing at -20°C (FRN), FRN followed by defrosting (FRNdef), shock-freezing with liquid N and freeze-drying (FDN), freeze-drying (FD), freeze-drying and storage for three weeks (FD3W) and six months (FD6M). MA metaproteomics workflow was used to analyse the effects of treatments C, FR, FRN, FD, and FDN on to study changes in microbial community composition and functionactivity.

INSTRUMENT(S):

ORGANISM(S): Ovis Aries

TISSUE(S): Feces

SUBMITTER: Jana Seifert

LAB HEAD: Prof. Jana Seifert

PROVIDER: PXD060439 | Pride | 2026-03-27

REPOSITORIES: Pride

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			Action	DRS
	ExperimentalDesign.tsv	Tabular
	QuantifiedPeaks.tsv	Tabular
	QuantifiedPeptides.tsv	Tabular
	QuantifiedProteins.tsv	Tabular
	QuantifiedProteinsRazor.tsv	Tabular

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Project description:Purpose: The goal of this study was to determine the microRNA (miRNA) content of extracellular vesicles (EVs) derived from murine mesenchymal stem cells (mMSC), and evaluate reproducibility among distinct EV productions. We also aimed at assessing the effect of freeze-drying on EV miRNA content, by performing sequencing on freeze-dried EVs and calculating statistical difference between unmodified and freeze-dried EVs. Methods: mMSC-derived EVs were obtained from mMSC in culture in reduced serum medium Opti-MEM by differential centrifugation, with a final step at 100,000 g for 110 min at 4°C. EV pellets (freeze-dried (n=3) or not (n=2)) were resuspended in Qiazol lysis buffer and RNA was extracted following RNeasy Micro kit. cDNA libraries for sequencing were prepared using the TruSeq Small RNA Sample Preparation Kit. Amplified cDNA constructs were purified on 6 % PAGE gel and DNA molecules corresponding to 15–50 nucleotide transcripts were excised, eluted from gel, and concentrated. Image analyses and base calling were performed using the HiSeq Control Software and Real-Time Analysis component (Illumina). Before statistical analysis, genes with less than 15 reads (cumulating all the analysed samples) were filtered out. Differentially expressed miRNA were identified using three Bioconductor packages: edgeR, DESeq and DESeq2. Results: Considering miRNAs detected with at least 5 counts (in terms of normalised counts), 339 miRNAs were identified and miRNA content was highly conserved among the two batches tested, with 237 miRNAs out of the 339 present in both batches (70%). Statistical analysis did not evidence statistical difference between unmodified EVs (n=2) and freeze-dried EVs (n=3) (DESeq2, p<0.05). No statistical difference was found using other Bioconductor packages DESeq and edgeR. These results indicated conservation of miRNA content following freeze-drying. Conclusion: mMSC-EV miRNA content was comparable between the two EV productions analysed, indicating reproducibility. Some of the miRNAs identified were consistent with previously published results on MSC-derived EVs. Freeze-drying conserved miRNA content.

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Sheep faeces preservation affects microbiome activity

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