The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isolated from cow rumen content stored under different conditions was conducted. The influence of rumen fluid treatment (cheesecloth squeezed, centrifuged, filtered), storage temperature (RT, -80 °C) and cryoprotectants (PBS-glycerol, ethanol) on quality and quantity parameters of extracted DNA was evaluated by bacterial DGGE analysis, real-time PCR quantification and metabarcoding approach using high-throughput sequencing. Samples clustered according to the type of extracted DNA due to considerable differences between iDNA and eDNA bacterial profiles, while storage temperature and cryoprotectants additives had little effect on sample clustering. The numbers of Firmicutes and Bacteroidetes were lower (P < 0.01) in eDNA samples. The qPCR indicated significantly higher amount of Firmicutes in iDNA sample frozen with glycerol (P < 0.01). Deep sequencing analysis of iDNA samples revealed the prevalence of Bacteroidetes and similarity of samples frozen with and without cryoprotectants, which differed from sample stored with ethanol at room temperature. Centrifugation and consequent filtration of rumen fluid subjected to the eDNA isolation procedure considerably changed the ratio of molecular operational taxonomic units (MOTUs) of Bacteroidetes and Firmicutes. Intracellular DNA extraction using bead-beating method from cheesecloth sieved rumen content mixed with PBS-glycerol and stored at -80 °C was found as the optimal method to study ruminal bacterial profile.

Institute of Animal Physiology and Genetics CAS v v i Vídeňská 1083 Prague 14220 Czech Republic

Laboratoire d'Ecologie Alpine CNRS 2233 rue de la Piscine 38041 Grenoble France

MTT Agrifood Research Finland Animal Genomics FI 31600 Jokioinen Finland

MTT Agrifood Research Finland Animal Nutrition and Nutrigenomics FI 31600 Jokioinen Finland

Rowett Institute of Nutrition and Health University of Aberdeen Bucksburn Aberdeen AB21 9SB United Kingdom

Università Cattolica del Sacro Cuore Piacenza Italy

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