Effects of preservation method on canine (Canis lupus familiaris) fecal microbiota

Abstract

Studies involving gut microbiome analysis play an increasing role in the evaluation of health and disease in humans and animals alike. Fecal sampling methods for DNA preservation in laboratory, clinical, and field settings can greatly influence inferences of microbial composition and diversity, but are often inconsistent and under-investigated between studies. Many laboratories have utilized either temperature control or preservation buffers for optimization of DNA preservation, but few studies have evaluated the effects of combining both methods to preserve fecal microbiota. To determine the optimal method for fecal DNA preservation, we collected fecal samples from one canine donor and stored aliquots in RNAlater, 70% ethanol, 50:50 glycerol:PBS, or without buffer at 25°C, 4°C, and -80°C. Fecal DNA was extracted, quantified, and 16S rRNA gene analysis performed on days 0, 7, 14, and 56 to evaluate changes in DNA concentration, purity, and bacterial diversity and composition over time. We detected overall effects on bacterial community of storage buffer (F-value= 6.87, DF= 3, P<0.001), storage temperature (F-

Variations of gut microbiome profile under different storage conditions and preservation periods: A multi-dimensional evaluation

“…Similarly, contradictory results are also observed in the use of RNAlater. Study shows that fecal samples preserved with RNAlater closely resembles that of fresh samples (20), but contrary results are observed in another study, in which RNAlater-preserved samples have the least similarity in microbial composition and abundance with fresh samples (24). In addition, significant differences are also observed in microbial profile of identical samples that processed and sequenced at two research centers (25, 26).…”

“…The genomic DNA of all samples was extracted by one experimenter with same protocol in our current study. Furthermore, many studies evaluated the effectiveness of preservation methods by using −80 °C or GT reagents stored samples as controls, whereas the efficacy of these preservation conditions are still inconclusive (20). Thus, a part of fecal sample divided from the homogenized mixture on-site was used as the fresh control for comparison with other storage conditions.…”

“…Thus, a part of fecal sample divided from the homogenized mixture on-site was used as the fresh control for comparison with other storage conditions. Stabilization of fecal samples with ethanol is an easy and economical way, and therefore different concentrations of ethanol were used for fecal sample preservation in previous reports including 100% ethanol for spider monkey (24), 95% ethanol for human or dog (22), and 70% ethanol for canine fecal sample storage (20). However, inconsistent results were observed in different studies.…”

“…Generally, immediate freezing at −80 °C is supposed to be the gold standard for most biological samples including feces for microbiome study (18, 19). However, obvious alteration in microbial community is also observed in samples frozen at −80 °C compared to that of fresh samples, suggesting that −80 °C might not be the most optimal method for fecal sample storage (20). Moreover, immediate freezing at −80 °C is usually unfeasible in many cases for field studies, or at the circumstance where the fecal samples are collected at home by patients themselves.…”

“…Song et al find that the preserving effect of 95% ethanol is comparable with that of FTA cards or OMNIgene Gut kit at ambient temperature, but strongly caution against the use of 70% ethanol for fecal sample preservation (22). Contrarily, Horng et al report that the microbial composition of samples preserved with 70% ethanol or RNAlater closely resembles that of fresh samples, and they suggest that 70% ethanol is the best method for preserving canine fecal samples (20). Similarly, contradictory results are also observed in the use of RNAlater.…”