Academic supervisors Senior researcher, Nuria Canibe, PhD Department of Animal Science, Faculty of Science and Technology, Aarhus University, 8830, Tjele, Denmark. Senior researcher Emeritus, Bent Borg Jensen, PhD Department of Animal Science, Faculty of Science and Technology, Aarhus University, 8830, Tjele, Denmark. Assessment committee Associated Professor Jan Værum Nørregaard, PhD (Chair) Department of Animal Science, Faculty of Science and Technology, Aarhus University, Tjele, Denmark. Professor Jan Erik Lindberg, PhD Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, Uppsala, Sweden. Professor Ulrike Weiler, PhD Institute of Animal Husbandry and Animal Breeding, Behavioral Physiology of Farm Animals, University of Hohenheim, Stuttgart, Germany I Preface This PhD thesis is submitted to the Faculty of Science and Technology, Aarhus University, Denmark, to fulfil the requirements of the PhD degree. It represents my work conducted at Department of Animal Science, in the research group of Immunology and Microbiology, from June 2013 to November 2016, interrupted by a maternity leave, giving in a total period of 3.5 years. This study has been supervised by the main supervisor, senior researcher Nuria Canibe, and the co-supervisor, senior researcher emeritus Bent Borg Jensen. The work was carried under the project ‘Organic pig production without castration’ that is part of the Organic RDD program, which is coordinated by International Centre for Research in Organic Food Systems, ICROFS. It was founded by Danish AgriFish Agency, Ministry of Food, Agriculture and Fisheries (Project 3405-10-OP-00134). The PhD student was supported by a scholarship from the China Scholarship Council (CSC). II Acknowledgments Foremost, I would like to express my sincere appreciation and thanks to both of my supervisors Nuria Canibe and Bent Borg Jensen for their continuous support of my study and research, for their patience, motivation, enthusiasm, and unparalleled knowledge. Without their consistent guidance, it would not be possible to complete this project. I would also like to thank my master supervisor Jiufeng Wang who recommended me to join this group. Besides my supervisors, I would like to thank all my co-authors for their insightful comments and contributions to my papers. I must say a special thank you to Dr Ole Højberg and Dr Samantha Joan Noel, for their guidance, support and encouragment. My sincere thanks also goes to all technicians: Mona Dinsen, Karin Durup, Trine Poulsen, Thomas Rebsdorf, Helle Handll, Kasper Poulsen, Britta Poulsen for skillful technical assistance in carrying out my experiments. I also appreciate help from Henry Johannes Høgh Jørgensen for assisting to perform the catheter surgery. I appreciate my previous and present officemates: Morten Poulsen, Grethe Venås Jakobsen, Zhigang Zhu, joyfully accompany and support. A warm thanks to all my colleagues in the group of Immunology and Microbiology for creating a harmonious environment. In particular, I would like to thank Ann-Sofie Riis Poulsen and Marlene Fredborg, for their willingness to share and help. And a big thanks to our secretory Mette Graves Madsen for all the assistance. Many thanks go to Nørresø kollegiet residents, the Chinese community in Foulum and the International club. Without them, my life in Denmark would not be that colorful. I would also like to thank all of my friends for supporting and understanding. Last but not the least, I am thankful to my family: my selfless parents for being babysitting in Denmark, my talented sister for backing, and my dear husband Jin for supporting me spiritually throughout my PhD and my life in general. In particular, I am grateful to my little daughter Shirley for reminding me that I am a mother besides a PhD. Xiaoqiong Li/ Foulum, December 2016 III List of scientific publications and manuscripts Paper I: Li X, Jensen RL, Højberg O, Canibe N, Jensen BB. 2015. Olsenella scatoligenes sp. nov., a 3-methylindole- (skatole) and 4-methylphenol- (p-cresol) producing bacterium isolated from pig faeces. International Journal of Systematic and Evolutionary Microbiology 65:1227–33. Paper II: Li X, Højberg O, Noel SJ, Canibe N, Jensen BB. 2016. Draft Genome Sequence of Olsenella scatoligenes SK9K4T, a Producer of 3-Methylindole (Skatole) and 4-Methylphenol (p-Cresol), Isolated from Pig Feces. Genome Announcements 4:e00042-16. Paper III: Li X, Marshall IPG, Schreiber L, Højberg O, Canibe N, Jensen BB. 2016. Draft Genome Sequence of Megasphaera sp. Strain DJF_B143, an Isolate from Pig Hindgut Unable to Produce Skatole. Genome Announcements 4:e00007-16. Paper IV: Li X, Jensen BB, Højberg O, Noel SJ, Canibe N. Development of a species-specific Taqman-MGB real-time PCR assay to quantify Olsenella scatoligenes in pigs offered a chicory root-based diet (planned to be submitted to Applied Microbiology Biotechnology) Paper V: Li X, Højberg O, Canibe N, Jensen BB. 2016. Phylogenetic diversity of cultivable butyrate-producing bacteria from pig gut content and feces. Journal of Animal Science 94:377–381. Paper VI: Li X, Jensen BB, Canibe N. Effect of chicory roots and exogenous butyrate on skatole production and gut microbiota of entire male pigs (planned to be submitted to Applied and Environmental Microbiology). IV List of abbreviations CYP Cytochromes P450 DF Dietary fiber DGGE Denaturing gradient gel electrophoresis DP Degree of polymerization FISH Fluorescence in situ hybridization FOS Fructo-oligosaccharides GI Gastrointestinal GnRH Hypothalamic gonadotropin-releasing hormone HTS High-throughput sequencing IAA Indole acetic acid IPA Indole propionic acid ITF Inulin-type fructans MGB Minor groove binder NGS Next-generation sequencing NSP Non-starch polysaccharides OTU Operational taxonomic unit QIIME Quantitative Insights Into Microbial Ecology qPCR Quantitative real-time PCR RPS Raw potato starch RS Resistant starch SCFA Short-chain fatty acid TRP L-tryptophan T-RFLP Terminal restriction fragment length polymorphism WGS Whole-genome shotgun V Summary Skatole, a cytotoxic and malodorous compound, is the main causer of boar taint, which is an offensive odor and flavor released upon heating meat from some pubertal or sexually mature male pigs. Due to animal welfare concerns, there is an increasing desire to stop surgical castration within the European Union by 2018, turning boar taint into a challenge in pig production. Skatole is produced by microbial fermentation of L-tryptophan (TRP) in the hindgut. The production involves a two-step process, in which the precursor TRP is first deaminated to indole acetic acid (IAA), which is then further decarboxylated to skatole. Difficulties arise in controlling boar taint, due to the lack of knowledge on the specialized gastrointestinal skatole-producing bacteria. Skatole levels can be reduced by feeding certain dietary fibers (DF), making this strategy an alternative to surgical castration. Dietary fiber sources rich in inulin-type fructans (e.g. chicory roots and Jerusalem artichoke) are the most effective in reducing skatole formation as seen so far. However, the exact mechanism underlying the skatole reducing effect of DF remains unclear. Hence, the objective of this PhD thesis was to screen and characterize the skatole-producing bacteria inhabiting the hindgut of pigs, their metabolism, genome, quantification, and to elucidate the mode of action behind the reducing effect of DF on skatole production in the hindgut of pigs. We screened 122 OTUs, which represent 2678 isolates from the GI-tract of pigs for their ability to produce skatole. Results showed that only one strain, O. scatoligenes SK9K4, had the capacity to produce skatole from IAA, but not from TRP. The 2.47 Mbp draft genome sequence of O. scatoligenes SK9K4T confirmed that it possesses the tyrosine degradation IV (to p-cresol) pathway, and offered the possibility to identify candidate genes for skatole production by comparative genomic analysis. Use of Taqman-MGB qPCR, enabled specific enumeration of O. scatoligenes, and showed that this bacterium only accounted for less than 0.01% of the total bacterial population in the pig hindgut. High levels of dietary chicory roots promoted O. scatoligenes proliferation, and selectively promoted the growth of specific butyrate-producing bacteria such as Megasphaera elsdenii, but not a general increase in the population of butyrogenic bacteria in pig gut. This indicated that the known effect of chicory roots for reducing skatole production is not by inhibiting the growth of the skatole-producing bacteria or by a general increasing of the butyrate-producing bacteria. Intracecal infusion of butyrate alone did failed to reduce skatole formation in the pig hindgut. In conclusion, cultivable skatole-producing bacteria in the pig GI-tract seem to be limited to one VI species, i.e., O. scatoligenes. The known effect of chicory roots for reducing skatole production is neither by inhibiting the growth of this skatole-producing bacteria in the hindgut, nor by inhibition of cell apoptosis by butyrate, but is more likely due to more TRP incorporated into microbial biomass. VII Resumé (Danish summary) Skatol, en cytotoksisk og ildelugtende forbindelse, er den vigtigste komponent af ornelugt, en ubehagelig lugt og smag der frigives ved opvarmning af kød fra specielt seksuelt modne hangrise. Kirurgisk kastration af hangrise er en almindelig