Evaluation of microbial signaling in the small intestine of weaned piglets under different nutritional regimes

The weaning of piglets is often accompanied by intestinal dysbiosis. Up to now, very little is known about how different health conditions affect the signaling chains and absorption of fatty acids in the intestine. Fatty acids play an important role in the signal transduction of inflammatory pathways. To counteract against this, additives, like organic acids, are added to the weaning feed. In this bachelor thesis the additives were selected with the aim of creating different homeostatic conditions. The aim of this thesis is to investigate 1) to what extent the gene expression of signal transduction and absorption of fatty acids in the duodenum and ileum of weanling piglets differs; and 2) how different feed additives influence the expression of FFAR1, FFAR2, FFAR3, FFAR4, MCT1, SMCT1, SMCT2, HCAR1, PPARG, AMPK, FASN and VDR in the duodenum and ileum. Two different feed additives were analysed: 0.5% benzoic acid, 3% carboxymethylcellulose and a combination of both, which were compared to a control diet. It was hypothesized that the benzoic acid leads to a reduced signal transduction by decreasing microbial activity. Furthermore, it was assumed that the animals, which were fed with carboxymethylcellulose show an opposite effect. The experiment was performed with 48 weaned pigs in two replicate batches. In each replicate batch the samples of the duodenum and Ileum were taken between the 39th and the 42nd day of life. The piglets received the experimental rations from the 33rd day of life. The samples of the duodenum and the ileum were pulverized with a mortar and a pestle using liquid nitrogen. The powder obtained served as the starting material for extracting the RNA. The concentration as well as the RNA Integrity Number (RIN) was determined. The RNA extraction was repeated if the RIN value was below 8. The RNA was then converted into cDNA, in order to be able to carry out the qPCR afterwards. The relative gene expression was calculated by using the 2−ΔΔCt-analysis. It was first normalized to the most stably expressed reference genes (ACTG and GAPDH). Subsequently, the expression was normalized to the animal with the highest gene expression. The statistical analysis was done by using an ANOVA and a mixed procedure was performed using the SAS-Software. The significance threshold was a p-value<0.05. A trend was present at a p-value between 0.05 and 0.1. The findings showed significant differences in the gene expression between the two intestinal segments. When considering the relative gene expression, the genes FFAR4, MCT1, SMCT1, PPARG, AMPK, FASN and VDR were higher expressed in the duodenum than in the ileum. All of the other genes, with the exception of HCAR1, showed a higher expression in the ileum. The absolute gene expression showed differences for the expression of the genes between the two intestinal sections. MCT1, SMCT1 and FASN were more expressed in the duodenum than in the ileum. All the other genes were more expressed in the ileum. The differences in the expression can be explained by the normalisation on the reference genes. With the exception of FASN in the duodenum none of the genes examined showed significant changes in gene expression as a result of the various feed additives. The benzoic acid caused a stronger expression of FASN in the duodenum compared to the diets without the benzoic acid. However, in the ileum this effect could not be seen. The sex of the animals only showed a significant influence on the expression of FFAR2 and MCT1 in the ileum. In conclusion, results demonstrated that the gene expression related to FA signalling and absorption differs significantly in the analysed intestinal sections. Differences between absolute and relative gene expression could be also identified. When examining the influence of feed additives, only FASN was significantly influenced by benzoic acid. With the exception of FFAR2 and MCT1, there was no significant influence of the sex of the animals on the expression of the genes.