Key words: 16S rRNA gene sequencing, high-grain feeding, hindgut, metagenomics, microbiome, rumen Introduction In recent years, the potential for milk production of dairy cows has increased substantially throughout the world. In order for these cows to meet their potential, high-energy diets must be fed. This is most often achieved by feeding more concentrates, especially grain, and less forages. However, these diets can adversely affect the microbiota of the diges- tive tract, affecting both the composition and functionality of microbiota, and potentially lead to colonization of opportunistic pathogens (Russell and Rychlik, 2001; Plaizier et al., 2008; Krause et al., 2013). Cows rely on their symbiosis with the micobiota in their rumen and intestines, as these microbiotas allow cows to digest fber, convert non-protein nitro- gen into protein, synthesize vitamins, and break down toxic compounds in digesta (NRC, 2001; Russell and Rychlik, 2001; Krause et al., 2013). Hence, adverse conditions for gut microbiota affect the health, produc- tion, and welfare of cows. Until recently, the technology was not available to comprehensively monitor the composition and functionality of micro- biota, as many microorganisms cannot be cultured, and quantitative PCR techniques do not target all microorganisms. Recent advances in sequenc- ing technologies offer rapid low-cost molecular-based methodologies that can investigate microbial communities as a whole (Krause et al., 2013). These new techniques are either based on high-throughput sequencing of hypervariable regions of highly conserved and universal 16S rRNA genes for bacterial and archaeal communities (Woese and Fox, 1977; Pace et al., 1985), 18S or the internal transcribed spacer (ITS) regions of rRNA genes for fungal and protozoal communities (Firkins and Yu, 2015), or massive shotgun sequencing of total DNA (metagenomics) or RNA (metatran- scriptomics) from microbial communities (Desai et al., 2012). These methodologies can be used to determine changes in the micro- bial community composition and function under high-grain feeding and identify strategies to reduce the impact of high-grain diets on the micro- biota of the digestive tract of high-yielding dairy cows, and, therefore, prevent poor gut health. Microbiota in the Ruminant Digestive Tract The digestive tract of ruminants is an immunologically active organ system, which is constantly exposed to a multitude of endogenous and ex- ogenous stimuli. The gastrointestinal (GI) tract is also home to a complex and diverse ecosystem of microbes known as the microbiota or microbiome. The number of bacterial species present in the GI tract of ruminants varies depending on the diet, feeding strategy, and geographical location and has been estimated to be more than 5,000 (Henderson et al., 2015). It is important to note that when we describe the microbiome using omics methodologies, where microorganisms are not directly observed/assessed, we often use “op- erational taxonomic units” (OTUs) instead of “species.” In this context, a unique OTU is defned as a cluster of sequence reads with a given similarity that is expected to be assigned to a taxonomical level; for instance, sequences with 97% similarity are expected to approximately correspond to species. Different ecological measures, such as richness, abundance, evenness, and diversity, are used to describe and compare microbiotas among ani- mals and across treatments (Caporaso et al., 2010; Gotelli and Colwell, 2010). Whereas richness refers to the number of different OTUs that are present in a given community, evenness and diversity also take into ac- count the abundances of these OTUs Gut microorganisms differ in their functionality and their ability to use fractions of the substrate resources in the digestive tract (Levine and D’antonio, 1999; Henderson et al., 2015). Hence, a high microbiota rich- ness, evenness, and diversity is considered benefcial, as this enhances the stability of the microbiota, especially during nutritional challenge conditions, and allows it to use limiting resources more effciently (Russell and Rychlik, 2001; Ley et al., 2006b). A decrease in richness and diversity has been re- ported in humans with Crohn’s disease (Dicksved et al., 2008; Walker et al., 2011) and type 1 and type 2 diabetes (Giongo et al., 2011). In ruminants, a number of metabolic disorders, such as subacute and acute ruminal acidosis, Effects of grain feeding on microbiota in the digestive tract of cattle E. Khafipour,*† S. Li,* H.M. Tun,* H. Derakhshani,* S. Moossavi,† and J.C. Plaizier* * Department of Animal Science, University of Manitoba, Winnipeg, MB, Canada † Department of Medical Microbiology, University of Manitoba, Winnipeg, MB, Canada © Khafpour, Li, Tun, Derakhshani, Moossavi, and Plaizier. doi:10.2527/af.2016-0018 Implications • Culture-independent high-throughput sequencing methodologies have recently provided comprehensive insights into microbial communities in the digestive tract of cattle. • Although the composition and the functionality of the microbiota in the digestive tract of cattle are considered robust, high-grain feeding reduces the richness, diversity, and functionality of these microbiota, and thereby affects animal health and production. • Cattle vary in their susceptibility to these adverse effects of high- grain feeding. • Adverse effects of high-grain feeding to cattle can be attenuated, but not prevented, by the use of supplements, such as buffers, yeasts, yeast culture products, direct-fed microbials, and probiot- ics, as well as by microbiota engineering. 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