fmicb-09-02144 September 11, 2018 Time: 18:48 # 1 ORIGINAL RESEARCH published: 13 September 2018 doi: 10.3389/fmicb.2018.02144 Rabbit Microbiota Changes Throughout the Intestinal Tract María Velasco-Galilea1*, Miriam Piles1, Marc Viñas2, Oriol Rafel1, Olga González-Rodríguez1, Miriam Guivernau2 and Juan P. Sánchez1 1 Animal Breeding and Genetics, Institute for Food and Agriculture Research and Technology (IRTA), Barcelona, Spain, 2 Integral Management of Organic Waste, Institute for Food and Agriculture Research and Technology (IRTA), Barcelona, Spain To gain insight into the importance of carefully selecting the sampling area for intestinal microbiota studies, cecal and fecal microbial communities of Caldes meat rabbit were characterized. The animals involved in the study were divided in two groups according to the feed intake level they received during the fattening period; ad libitum (n = 10) or restricted to 75% of ad libitum intake (n = 11). Cecum and internal hard feces were sampled from sacrificed animals. Assessment of bacterial and archaeal populations was performed by means of Illumina sequencing of 16S rRNA gene amplicons in a MiSeq Edited by: platform. A total of 596 operational taxonomic units (OTUs) were detected using QIIME David William Waite, University of Auckland, New Zealand software. Taxonomic assignment revealed that microbial diversity was dominated by Reviewed by: phyla Firmicutes (76.42%), Tenericutes (7.83%), and Bacteroidetes (7.42%); kingdom Huan Li, Archaea was presented at low percentage (0.61%). No significant differences were Lanzhou University, China Neil Ross McEwan, detected between sampling origins in microbial diversity or richness assessed using Aberystwyth University, two alpha-diversity indexes: Shannon and the observed number of OTUs. However, the United Kingdom analysis of variance at genus level revealed a higher presence of genera Clostridium, *Correspondence: Anaerofustis, Blautia, Akkermansia, rc4-4, and Bacteroides in cecal samples. By María Velasco-Galilea [email protected] contrast, genera Oscillospira and Coprococcus were found to be overrepresented in feces, suggesting that bacterial species of these genera would act as fermenters at Specialty section: the end of feed digestion process. At the lowest taxonomic level, 83 and 97 OTUs This article was submitted to Microbial Symbioses, in feces and cecum, respectively, were differentially represented. Multivariate statistical a section of the journal assessment revealed that sparse partial least squares discriminant analysis (sPLS- Frontiers in Microbiology DA) was the best approach for this purpose. Interestingly, the majority of the most Received: 10 May 2018 Accepted: 21 August 2018 discriminative OTUs selected by sPLS-DA were found to be differentially represented Published: 13 September 2018 between sampling origins in univariate analysis. Our study provides evidence that the Citation: choice of intestinal sampling area is relevant due to important differences in some Velasco-Galilea M, Piles M, Viñas M, taxa’s relative abundance that have been revealed between rabbits’ cecal and fecal Rafel O, González-Rodríguez O, Guivernau M and Sánchez JP (2018) microbiota. An appropriate sampling intestinal area should be chosen in each microbiota Rabbit Microbiota Changes assessment. Throughout the Intestinal Tract. Front. Microbiol. 9:2144. Keywords: gut microbiota, fecal microbiota, cecal microbiota, feed restriction, meat rabbit, paired analysis, doi: 10.3389/fmicb.2018.02144 multivariate approaches, 16S Illumina sequencing Frontiers in Microbiology| www.frontiersin.org 1 September 2018| Volume 9| Article 2144 fmicb-09-02144 September 11, 2018 Time: 18:48 # 2 Velasco-Galilea et al. Domestic Rabbit Gut Microbiota INTRODUCTION of the animal’s gastrointestinal tract that could alter its microbial composition. Microbial populations that inhabit animals’ gastrointestinal tract constitute their microbiota: a complex ecosystem, able to autoregulate its own homeostasis. It is well known that a MATERIALS AND METHODS mammal’s intestinal microbiome plays a very important role in metabolic, nutritional, physiological, and immunological Experimental Design and Sampling processes (Flint et al., 2012) but also in farm animal’s The sampling materials from animals used in this study came productivity (Heinrichs and Lesmeister, 2005; Drouilhet et al., from an experiment conducted at the Institute for Food and 2016). A symbiotic relationship is therefore established between Agriculture Research and Technology (IRTA) between July 2012 the host and its intestinal microbiota. The emergence of and July 2014. This experiment was developed to estimate the next generation sequencing (NGS) techniques together with effect of the interaction between the genotype and the feeding an increasing reliability of reference taxonomic databases such regime on growth, feed efficiency, carcass characteristics, and as SILVA (Yilmaz et al., 2013), RDP (Wang et al., 2007), or health status of the animals. Toward this aim, 7,864 animals Greengenes (McDonald et al., 2012) have allowed a deeper from Caldes line (Gómez et al., 2002), selected since the 1980’s knowledge of the influence that intestinal microbiome exerts on to increase the average daily gain during the fattening period host animals. (32–66 days of age), were controlled since weaning. Animals In the case of the rabbit, the physicochemical properties were housed in 969 collective cages, with a surface of 0.38 m2, of its gastrointestinal tract (near neutral pH, high humidity ◦ containing eight rabbits each one. All animals in this experiment and stable temperature around 35–40 C) promote the rapid were bred under the same management conditions and fed growth of mutualistic microbiota while the animal gets the with the same standard pellet diet supplemented with antibiotics bacterial fermentation end-products of some materials that (oxytetracycline, valnemulin, and colistin), except during the cannot be degraded by the host on its own (Mackie, 2002). last fattening week, when an antibiotic free food was provided. In these conditions, rabbit intestinal microbiota contains 100– During the 5 weeks that the fattening period lasted, food was 1000 billions of microorganisms per gram covering over 1000 supplied once per day in a feeder with three places. Details of food different species, predominating kingdom Bacteria over archaeal composition can be found in Table 1. Water was also provided populations (Combes et al., 2011). Despite the demonstrated ad libitum during the whole fattening period. existence of active microbial populations in proximal and distal The animals were under two different feeding regimes: (1) segments of rabbit gastrointestinal tract (Gouet and Fonty, 1979), ad libitum (V) or (2) restricted (R) feeding to 75% of the cecum is the main fermenter organ. For this reason, most studies ad libitum feed intake. The amount of food provided to the that aimed to unravel rabbit’s intestinal microbiota have been animals under R feeding regime in a given week for each batch focused on the characterization of cecal microbial communities was obtained as 0.75 times the average feed intake of kits on (Abecia et al., 2005; Kušar and Avguštin, 2010; Bäuerl et al., V from the same batch during the previous week, plus 10% 2014). Cecal microbiota of rabbit and other lagomorph species corresponding to the estimated increase of feed intake as the is dominated by phylum Firmicutes while cecal microbiota of animal grows. rodents, a relatively close mammalian order, is dominated by Kits were randomly assigned to one of these two feeding phylum Bacteroidetes (Li et al., 2017). regimes after weaning (32 days of age). They were categorized In other monogastric livestock species, such as chicken and into two groups according to their size (“big” if body weight pig, previous studies have characterized the differences between at weaning was greater than 700 g or “small” otherwise) in their cecal and fecal microbiotas (He et al., 2016; Oakley and order to obtain homogenous groups regarding animal size within Kogut, 2016; Fang et al., 2017). Crowley et al.(2017) compared the microbial composition from different organs of the digestive tract (stomach, jejunum, cecum, appendicular cecum, proximal TABLE 1 | Feed composition on a wet basis. colon, distal colon, and rectum) in wild rabbits and they found that the different physicochemical properties of each Component Amount compartment restrict or promote the growth of specific microbial populations. However, little is known about the differences in Crude fiber (%) 18.70 the composition of the microbial communities that inhabit the Crude protein (%) 15.02 domesticated rabbit cecum and feces. Ashes (%) 8.97 The objective of this study was to characterize and compare Ether extract (%) 3.28 the microbial communities of hard feces and cecum content Oxytetracycline (mg/kg) 400 Valnemulin (mg/kg) 30 collected from two groups of animals from a meat rabbit line Colistin (mg/kg) 100 fed with different intake levels. Our results will help establish whether feces could be considered a proxy indicator to assess The average daily feed intake in one ad libitum cage was 0.17 kg/day/rabbit composition and diversity of intestinal microbiota. This will be which implies 66.48 mg/rabbit of oxytetracycline, 4.99 mg/rabbit of valnemulin and 16.62 mg/rabbit of colistin. The average daily feed intake in one restricted particularly important for those studies that require a monitoring cage was 0.13 kg/day/rabbit which