Effect of Freezing Conditions on Fecal Bacterial Composition in Pigs.

Barbara Metzler-Zebeli,Peadar Lawlor,Qendrim Zebeli,Elizabeth Magowan

doi:10.3390/ani6030018

Abstract

Simple SummaryStorage of gut samples may affect the extractability of intact DNA and analyzed bacterial composition. In this study, we compared the DNA yield and the abundance of total bacteria and eight bacterial taxa when DNA was extracted from fresh fecal samples of pigs or from freeze stored samples with or without prior snap-freezing in liquid nitrogen. Results showed that the greatest differences in DNA yield and bacterial abundances were found when DNA was extracted from fresh feces compared to freeze stored fecal samples. 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.

Highlights

Investigating the composition of gut microbiota composition and correlating findings to specific physiological states has shown the importance of the bacterial community present in the gut as a regulatory factor in health and disease [1,2]
Our results demonstrated that freezing of pig feces prior to DNA extraction significantly reduced the resultant DNA yield, the absolute bacterial abundance and modified the bacterial profile when compared to immediate DNA extraction from fresh feces
Our data further indicated that the findings reported for the freeze storage effect on human stool samples can only be partly transferred to pig fecal samples

Summary

Introduction

Investigating the composition of gut microbiota composition and correlating findings to specific physiological states has shown the importance of the bacterial community present in the gut as a regulatory factor in health and disease [1,2]. It is well recognized that molecular-based approaches provide a broader understanding about the diversity and distribution of microbial species in the gut of pigs than culture-based approaches alone [2]. Approaches are widely used to quantify microbial taxa in gut digesta, feces and gut mucosa scrapings and to monitor pathogen and virulence factor abundance in feces of pigs undergoing treatment [4,5,6]. For all these techniques, it is obligatory that the phylogenetic composition of the extracted DNA reflect the original bacterial community composition [7].

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Methods

Results

Conclusion