title: Effect of Freezing Conditions on Fecal Bacterial Composition in Pigs.
abstract: Sample preservation and recovery of intact DNA from gut samples may affect the inferred gut microbiota composition in pigs. This study aimed to evaluate the effect of the freezing process and storage temperature prior to DNA extraction on DNA recovery and bacterial community composition in pig feces using quantitative PCR. Fresh fecal samples from six growing pigs were collected and five aliquots of each prepared: (1) total DNA extracted immediately; (2) stored at -20 °C; (3) snap frozen and stored at -20 °C; (4) stored at -80 °C; and (5) snap frozen and stored at -80 °C. Results showed that DNA yields from fresh fecal samples were, on average, 25 to 30 ng higher than those from the various stored samples. The DNA extracted from fresh samples had more gene copies of total bacteria and all targeted bacterial groups per gram feces compared to DNA extraction from frozen samples. Data presentation also modified the observed effect of freeze storage; as results for Lactobacillus group, Enterococcus spp., Streptococcus spp., Clostridium cluster IV, Bacteroides-Prevotella-Porphyromonas and Enterobacteriaceae showed the opposite effect when expressed as relative abundance, by being greater in freeze stored feces than in fresh feces. Snap freezing increased the relative proportion of Clostridium cluster IV by 24%. In conclusion, the freezing process affected DNA yield and bacterial abundances, whereas snap freezing and storage temperature had only little influence on abundances of bacterial populations in pig feces.
authors.affiliation: Department of Farm Animals and Veterinary Public Health, UniversityClinic for Swine, Vetmeduni Vienna, Veterinaerplatz 1, 1210 Vienna, Austria. barbara.metzler@vetmeduni.ac.at.
authors.forename: Barbara U
authors.initials: BU
authors.lastname: Metzler-Zebeli
authors.affiliation: Pig Development Department, Animal and Grassland Research and Innovation Centre, Teagasc, Moorepark, Fermoy, Co. Cork P61 C996, Ireland. peadar.lawlor@teagasc.ie.
authors.forename: Peadar G
authors.initials: PG
authors.lastname: Lawlor
authors.affiliation: Agri-Food and and Biosciences Institute, Large Park, Hillsborough, N. Ireland BT26 6DR, UK. Elizabeth.Magowan@afbini.gov.uk.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Magowan
authors.affiliation: Department of Farm Animals and Veterinary Public Health, Institute of Animal Nutrition and Functional Plant Compounds, Vetmeduni Vienna, Veterinaerplatz 1, 1210 Vienna, Austria. qendrim.zebeli@vetmeduni.ac.at.
authors.forename: Qendrim
authors.initials: Q
authors.lastname: Zebeli
keywords: DNA; bacterial composition; feces; freezing condition; pig; quantitative PCR