Development of gut microbiota in the pig modulation of bacterial communities by different feeding strategies

El objetivo de esta tesis fue el estudio de la microbiota gastrointestinal porcina, para mejorar el conocimiento existente de este complejo ecosistema y facilitar el desarrollo de nuevas estrategias para sustituir los antibioticos promotores del crecimiento. En la Prueba I, se desarrollo la PCR cuantitativa para cuantificar bacterias totales, lactobacilli y enterobacteria en digesta. Para validarla, los resultados obtenidos se compararon con los que se obtuvieron con metodos tradicionales La relacion lactobacilli:enterobacteria fue similar entre metodos. Los contajes para el total de bacterias y lactobacilli mostraron una correlacion significativa. Por ello, el metodo se considero como valido para cuantificar cambios bacterianos en el tracto gastrointestinal del cerdo. Con el fin de estudiar el establecimiento de la microbiota en el cerdo tras el destete, se diseno la Prueba II. En esta, 12 lechones de 6 camadas fueron divididos en un grupo control, el cual permanecio con la madre, y un grupo experimental el cual fue destetado y alimentado con una dieta comercial. Tras una semana, los animales fueron sacrificados. Hubo un descenso importante en el ratio lactobacilli:enterobacteria. Ademas, el analisis de similaridad de los perfiles obtenidos por t-RFLP, mostro una agrupacion separada de los grupos experimentales. Estos resultados confirman el destete como un punto critico en el establecimiento de la microbiota gastrointestinal. En la Prueba III, se utilizaron cerdos en crecimiento para estudiar la microbiota gastrointestinal y el potencial de la fibra para modificar este ecosistema. Para ello, 32 cerdos (15 ± 0.38 kg) se distribuyeron en 4 tratamientos: una dieta control, una dieta con maiz de mayor tamano de particula, una con pulpa de remolacha, y una cuarta dieta con salvado de trigo. Tras seis semanas, los animales fueron sacrificados. La tecnica del FISH se utilizo con el fin de describir los grupos bacterianos mayoritarios a lo largo del tracto gastrointestinal. En estomago, streptococci y lactobacilli fueron los grupos predominantes, mientras que en intestino grueso, el grupo de Bacteroides/Prevotella, clostridial cluster XIVa, IV, y ruminococci fueron los mas abundantes. Los resultados obtenidos por RFLP mostraron cambios en el perfil bacteriano dependiendo de la dieta administrada. Los animales que recibieron salvado de trigo mostraron una menor biodiversidad con unos perfiles mas similares entre animales. En la Prueba IV, se testaron 3 aditivos: avilamicina (como control positivo), butirato sodico y un extracto de plantas. Un total de 40 (18-22 dias) cerdos se distribuyeron en cuatro tratamientos: una dieta control, esta con 0.04% de avilamicina, con 0.3% de butirato sodico o con 0.03% de extracto de plantas. Despues de dos semanas los animales fueron sacrificados. La relacion lactobacilli:enterobacteria en ciego fue superior para los animales que recibieron el extracto de plantas. El RFLP mostro diferencias en el perfil bacteriano, agrupando los animales en funcion de la dieta administrada. La actividad bacteriana total medida como bases puricas tambien mostro diferencias entre dietas. Estos resultados podrian indicar que el efecto de los diferentes aditivos testados no se deberia a modificaciones en la composicion y actividad de la microbiota. En la Prueba V, una fuente comercial de mananoligosacaridos y de zinc organico, administrados por separado o conjuntamente fueron testados. Para ello, 128 cerdos (18-22 dias de vida) se distribuyeron en cuatro tratamientos: una dieta control, 0.2% de mananoligosacaridos, 0.08% de zinc organico o con ambos aditivos. Las dietas fueron administradas durante cinco semanas. Se observo una mejora en el indice de conversion para todo el periodo experimental cuando los dos aditivos se anadieron conjuntamente. Los mananoligosacaridos redujeron la enterobacterias en yeyuno. La adicion de zinc organico, tendio a incrementar el peso en vacio del ileon. Estos resultados sugieren diferentes mecanismos de accion de los aditivos. ______________________________________________ The main objective of this thesis was to study pig gut bacteria to improve our knowledge of this complex ecosystem as this could help in the development of new feed strategies to substitute antibiotics as growth promoters. In Trial I, real-time PCR was developed to quantify total bacteria, lactobacilli and enterobacteria in digesta samples. To validate it, results obtained were compared to those obtained by traditional methods. Values obtained by PCR and traditional methods showed a significant correlation for lactobacilli and total bacteria. Therefore, real-time PCR was considered a valid method to quantify microbial shifts in the gastrointestinal tract. To study pig gut microbiota establishment in the young pig after weaning, trial II was designed. Twelve pigs (20 days) from 6 litters were divided into a control group that remained with the sow and an experimental group that was weaned and fed a commercial post-weaning diet. After one week, the animals were sacrificed and samples from cecal digesta were taken. Total bacteria and biodiversity of the microbial ecosystem were similar between both experimental groups, although there was a decrease in the lactobacilli:enterobacteria ratio. Also, cluster analysis grouped animals in two different clusters. Results confirm weaning as a challenging point on the indigenous microbiota establishment. In Trial III, growing pigs were used to study pig gut microbiota and the potential of fiber to modify this ecosystem. A total of 32 pigs (15 kg) were distributed into four experimental diets: a control diet, a diet enriched in resistant starch by inclusion of coarse-ground corn, a diet with 8 % sugar beet pulp and a with 10 % wheat bran. After six weeks animals were sacrificed. FISH was applied to describe main bacterial groups along the gastrointestinal tract and to detect changes related to the diets. Streptococci and lactobacilli were predominant in the stomach whereas Bacteroides/Prevotella, clostridial cluster XIVa, IV, and ruminococci were predominant in the lower tract. The RFLP profiles showed changes in the bacterial profile related to diet, with pigs fed wheat bran showing the lowest biodiversity and also having the most similar patterns. Moreover, changes in fermentation activity were detected when short-chain fatty acids were measured. In Trial IV, three different additives were tested: avilamycin (as a positive control), sodium butyrate and a commercial plant extract. Forty weaned (18-22 d) pigs were distributed into four dietary treatments: a control diet, a diet with 0.04% avilamycin, with 0.3% sodium butyrate or with 0.03% plant extract mixture. After two weeks, the animals were sacrificed. There was an increase in the lactobacilli:enterobacteria ratio in the cecum of piglets fed with plant extracts. RFLP showed differences in microbial profile in jejunum digesta, with an increase in biodiversity with the different additives compared to control diet. Total microbial activity measured as purine bases showed differences between diets. Therefore, the effect of the different additives would be related to changes in the ecological structure and metabolic activity of the microbial community. In Trial V, mannan-oligosaccharides and organic zinc, offered alone or in combination, were evaluated to enhance performance, gastrointestinal health and immune response. A total of 128 early-weaned pigs were distributed into four dietary groups. For five weeks, animals received a control diet, a diet with 0.2 % mannan-oligosaccharides, with 0.08 % zinc or with both additives. An improvement in feed:efficiency was observed with both additives for the whole period. Mannan-oligosaccharides reduced enterobacteria counts in jejunum. Organic zinc tended to increase empty ileal weight, defined as the segment including the continuous Peyers patch, and crypt depths were lower in the animals offered both additives together. These results suggest different modes of action of the additives tested.