Caracterización del microbioma digestivo de una línea de conejo sometida a dos tratamientos alimentarios diferentes

Abstract

[EN] Feed restriction during the fattening period is a widespread method of management that is favorable for animal health because it reduces the risk of digestive problems such as mucoid enteropathy. Although feed restriction penalizes animal growth, it improves feed efficiency and reduces environmental impact, feeding costs and the use of antibiotics. Feeding restriction modifies gastrointestinal bacterial fermentation pattern and therefore it could change the micr obial composition and activity. The aim of this thesis is the characterization of caecal and faecal microbial communities that belong to two groups of meat rabbits which were fed with different intake levels during the fatting period, as well as to study t he existence of possible associations of some microorganisms with growth. Total DNA from caecum and faeces – belonging t 24 Caldes line rabbits; 11 fed ad libitum and 13 under restriction - was extracted with a bead - beating kit. A 411 bases fragment correspo nding to the hypervariable regions V4 and V5 of the 16S rRNA gene was amplified. Libraries of the 48 samples were generated following manufacturer’s instructions of kit Nextera® XT and then sequenced in parallel in a MiSeq Illumina sequencer. Readings gene rated were analyzed with the bioinformatic package QIIME yielding a table that includes the normalized abundances of 1.823 OTUs. A linear model was implemented to study the effects of feeding treatment, the origin of the sample and the interaction of both on the relative abundance of bacteria at different taxonomic levels: OTU, family and phylum. A principal coordinate analysis (PCoA) was performed from weighted Unifrac phylogenetic distance matrix. A linear model was also used to study the effect of microb ial composition on average daily gain (ADG) and weight at 31 days of life (P31). Finally, the effect of microbial diversity on ADG was studied with a linear model in which the explanatory factors were the feeding treatment and the regression on number of o bserved OTUs. The sequencing process generated 5.820.828 readings with large variability between samples (range=40.025 - 240.333) which after filtering yielded 2.195.158 contigs. Rarefaction curves obtained for diversity index showed no difference between th e levels of the combination feeding treatment - origin of the sample. This analysis detected four samples clearly separated from the rest. The taxonomic characterization of the samples revealed that the dominant phyla were Firmicutes (76,31%), Bacteroidetes (7,61%) and Tenericutes (7,52%); last one hadn’t been described in the literature before. A unique genus of the kingdom Archaea ( Methanobrevibacter ) was presented (0,65‰). Although the PCoA analysis did not show a clear pattern of separation between sample s depending on the combination of feeding treatment - origin; when the effect of the origin of the sample on the microbial composition was analyzed, significant differences were found for phyla Actinobacteria and Cyanobacteria ; for families Lachnospiraceae , Clostridiaceae , Coriobacteriaceae , an unknown family belonging to phylum Proteobacteria and an unknown family belonging to orde r YS2 ( Cyanobacteria ). When the effect of feeding regimen on the microbial composition was studied, significant differences were found for phyla Cyanobacteria and Tenericutes ; for an unknown family belonging to order YS2 and for an unknown xv family belonging to order RF39 ( Tenericutes ). PCoA showed that four atypical samples had a microbial composition different from the rest and evid enced significant differences for 6 phyla and 27 families. In addition, 9 OTU - classified within families Ruminococcaceae and Lachnospiraceae from phylum Firmicutes - were exclusive of them. Although the microbial composition of these 4 samples is not typic al of animals affected by enteropathy, one of them was diagnosed with enteric problems during the fattening period. The association study between microbial composition and average daily gain, showed a negative association for the family Mogibacteriaceae (o rder Clostridiales ) and a positive association for one OTU of unknown family belonging to the order Clostridiales . The study of association between ADG and the number of observed OTUs was only significant for the group of animals fed under restriction show ing a positive association between diversity and ADG. This study has tuned up MiSeq sequence technology for the study of microbial composition of caecal and faecal rabbit samples, we have also developed a pipeline based on the software QIIME. These achieve ments have allowed a preliminary study of the effect of feeding treatment and the origin of the samples on the microbial composition and its influence on g

[ES] Se pretenden determinar las diferencias en las poblaciones microbianas del aparato digestivo de una línea paternal de conejos sometida a dos regímenes de alimentación (a voluntad o restringida). Se considerarán muestras tanto del contenido cecal como de las heces, teniendo como objetivo aplicado la tesis el poder decidir qué tipo de muestra ofrece una información más valiosa para caracterizar las diferencias entre los tratamientos. De igual manera se trataría de asociar los distintos patrones microbiológicos encontrados con el crecimiento y la mortalidad de los animales.