Sampling, transportation and preservation of fecal and guts samples from the saiga population for 16S rRNA sequencing

Аннотация

Background and Aim. The collection, transport, and preservation of fecal and intestinal samples from wild animals are critical steps in microbiome research, particularly in studies of the saiga antelope microbiome using 16S rRNA sequencing to investigate complex host microbial interactions The aim of this study was to compare four methods for the transport and preservation of saiga fecal and intestinal samples in order to evaluate their effectiveness in maintaining microbial DNA integrity for subsequent 16S rRNA sequencing.

Materials and Methods. The research was conducted in the Bokeyorda State Nature Reserve, located in the West Kazakhstan region of Kazakhstan, with permission from the Ministry of Ecology and Natural Resources of the Republic of Kazakhstan. The animals were captured for study without harm to their health. Biological material in the form of feces from live saigas was collected using a non-invasive method into sterile test tubes. The autopsy of dead saigas was performed in clean field conditions using sterile instruments. All samples were immediately frozen in liquid nitrogen. Additionally, they were cooled to 4 °C, stored at room temperature, and preserved with RNAlater for subsequent analysis.

Results. A total of 37 samples were collected from two dead female saigas from different sections of the gastrointestinal tract, as well as fresh fecal samples and rectal swabs obtained from two live females and one male. Upon arrival at the laboratory, all samples were stored at −80 °C until the start of laboratory studies. The study demonstrated that sample storage conditions significantly affect the preservation of the saiga faecal microbiota structure. Samples stored at −80 °C showed the greatest stability in both alpha diversity and taxonomic composition of microbial communities. These findings are particularly relevant for microbiota research and RNA sequencing in remote and inaccessible regions of the saiga habitat.

Conclusion. The results suggest that in field conditions where strict cold-chain maintenance is not possible, particularly when sampling from wild and remote saiga populations, the use of liquid nitrogen or RNAlater provides an effective and practical alternative to rapid freezing.