When researchers performed intestinal microbiota analysis using fecal samples, sample preparation processes are not standardized as of now. In particular, contrary to lab conditions, there are various factors hard to be controlled when sampling livestock feces on the farm. In this study, we wanted to know the influences of sample preparation conditions on fecal microbiota. We designed an experiment considering various sample preparation conditions (sample origin: rectum and ground; transporting condition: lysis buffer-treated, dry ice, ice, and room temperature; delayed times to extract DNA: 0, 1, 6, and 24 h) that can occur during microbiota analysis using cattle feces. First, we investigated the influences of sample origin, and microbial diversity (observed OTUs, p<0.001) was significantly higher when fecal samples were obtained from the ground than from the rectum and the principal coordinates analysis plot showed that samples were divided into two groups by origin. When we investigated the influences of transporting and storage conditions, observed OTUs (p<0.05) was significantly higher when samples were transported at room temperature than other conditions, and microbiota was affected by transporting and storage conditions. Finally, we investigated the effects of delayed time before DNA extraction from frozen fecal samples on microbial composition. Although this factor did not have a significant influence on microbial diversity, multidimensional scaling revealed its impact on microbial composition. Our findings will contribute to establishing an optimal procedure for microbiota analysis using farm-housing livestock feces.

ABSTRACT1
Ⅰ. INTRODUCTION
Ⅱ. MATERIALS AND METHODS
    1. Sample preparation
    2. DNA extraction and sequencing
    3. Microbiota analysis
    4. Statistical analyses
Ⅲ. RESULTS
    1. Effects of sample origin on fecal microbiota
    2. Effects of transporting method and storage conditionon fecal microbiota
    3. Effects of delayed time before DNA extraction fromfrozen samples on microbial composition
Ⅳ. DISCUSSION
ACKNOWLEDGEMENTS
Ⅴ. REFERENCES

• 이상목(서울대학교 농생명공학부 대학원생) | Sang-Mok Lee (1Graduate Student, Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Science, Seoul National University, Seoul 08826, Korea)
• 한건구(서울대학교 농생명공학부 대학원생) | Geongoo Han (Graduate Student, Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Science, Seoul National University, Seoul 08826, Korea)
• 김은배(강원대학교 동물생명과학대학 동물응용과학과 교수) | Eun Bae Kim (Professor, Department of Applied Animal Science, College of Animal Life Sciences, Kangwon National University, Chuncheon 24341, Korea) Corresponding author
• 강상기(서울대학교 그린바이오 과학기술연구원 교수, 서울대학교 국제농업기술대학원 교수) | Sang-Kee Kang (Professor, Institute of Green-Bio Science and Technology, Seoul National University, PyeongChang 25354, Korea, Professor, Graduate School of International Agricultural Technology, Seoul National University, PyeongChang 25354, Korea) Corresponding author