Effects of some stable derivatives of butyrate used as feed additives on fish intestinal microbiota, histone modifications and the expression of genes related to epigenetic regulatory mechanisms and immune response.

Bacteria that inhabit the epithelium of the animals’ digestive tract provide the essential biochemical pathways for fermenting otherwise indigestible dietary fibers, leading to the production of short-chain fatty acids (SCFAs). Of the major SCFAs, butyrate has received particular attention due to its numerous positive effects on the health of the intestinal tract and peripheral tissues. Butyrate plays a major role in enhancing epithelial cell proliferation and differentiation and in improving the intestinal absorptive function. It has also potential immunomodulatory and anti-inflammatory properties in the intestine and may prevent colorectal cancer in humans. The mechanisms of action of butyrate are different; this four-carbon chain organic acid is a histone deacetylase inhibitor that play a predominant role in the epigenetic regulation of gene expression and cell function, therefore many of its mechanisms are related to its potent regulatory effect on gene expression. During the first year of PhD, my research activity was related to the study of the effects of dietary sodium butyrate on histone modifications and the expression of genes involved in epigenetic regulatory mechanisms and immune response in European sea bass (Dicentrarchus Labrax) fed a plant-based diet. Accordingly, the effects of butyrate used as a feed additive on fish epigenetics as well as its regulatory role in mucosal protection and immune homeostasis through impact on gene expression, were investigated. To meet the aims, seven target genes related to inflammatory response and reinforcement of the epithelial defense barrier [tnfα (tumor necrosis factor alpha) il1β, (interleukin 1beta), il-6, il-8, il-10, and muc2 (mucin 2)] and five target genes related to epigenetic modifications [dicer1(doublestranded RNA-specific endoribonuclease), ehmt2 (euchromatic histone-lysine-N-methyltransferase 2), pcgf2 (polycomb group ring finger 2), hdac11 (histone deacetylase-11), and jarid2a (jumonji)] were analyzed in fish intestine and liver. We also investigated the effect of dietary butyrate supplementation on histone acetylation, by performing an immunoblotting analysis on liver core histone extracts. Results of the eight-week feeding trial showed no significant differences in weight gain or Specific Growth Rate (SGR) in sea bass that received 0.2% sodium butyrate supplementation in the diet in comparison to control fish that received a diet without Na-butyrate. Dietary butyrate led to a two-fold increase in the acetylation level of histone H4 at lysine 8, but showed no effect on the histone H3 at Lys9. Moreover, two different isoforms of histone H3 that might correspond to the H3.1 and H3.2 isoforms previously found in terrestrial animals were separated on the immunoblots. The expression of four (il1 β, il8, irf1, and tnfα) out of seven analyzed genes related to mucosal protection and inflammatory response was significantly different between the two analyzed tissues but only il10 showed differences in expression due to the interaction between tissue and butyrate treatment. In addition, butyrate caused significant changes in vivo in the expression of genes related to epigenetic regulatory mechanisms such as hdac11, ehmt2, and dicer1. Statistical analysis by two-way ANOVA for these genes showed not only significant differences due to the butyrate treatment, but also due to the interaction between tissue and treatment. In the second year of my studies, I focused on a different fish species - gilthead sea bream (Sparus aurata), to investigate the effects of a specific combination of short- and medium-chain 1-monoglycerides on intestinal microbiome, gene expression, and fish growth performance. In aquaculture research, one important aim of the gut microbiota studies is to provide a scientific basis for developing effective strategies to manipulate gut microbial communities through the diet, promoting fish health and improving productivity. Currently, there is an increasing research interest towards the use of organic acids in commercial aqua-feeds, due to several beneficial effects they have on growth performance and intestinal tract’s health of farmed fish. Among organic acids, monoglycerides of short-chain fatty acids (SCFAs) and medium-chain fatty acids (MCFAs) have attracted particular research attention also for their bacteriostatic and bactericidal properties. Accordingly, the present study aimed to evaluate the potential beneficial effects of SCFA and MCFA monoglycerides, used as a feed additive, on fish growth performance, and intestinal microbiota composition. For this purpose, a specific combination of short- and medium-chain 1-monoglycerides (SILOhealth 108Z) was tested in 600 juvenile gilthead sea bream (Sparus aurata) of 60 g mean initial weight that were fed for 90 days with plant-based diets. Two isoproteic and isolipidic diets were formulated. The control fish group received a plantbased diet, whereas the other group received the same control feed, but supplemented with 0.5% of SILOhealth 108Z. The Illumina MiSeq platform for high-throughput amplicon sequencing of 16S rRNA gene and QIIME pipeline were used to analyse and characterize the whole microbiome associated with both thefeeds and S. aurata intestine. The number of reads taxonomically classified according to the Greengenes database was 394,611. We identified 259 OTUs at 97% identity in sea bream fecal samples; 90 OTUs constituted the core gut microbiota. Firmicutes, Proteobacteria and Actinobacteria represented the dominant phyla in both experimental groups. Among them, relative abundances of Firmicutes and Proteobacteria were positively and negatively affected by dietary SCFA monoglycerides supplementation, respectively. In summary, our findings clearly indicated that SILOhealth 108Z positively modulated the fish intestinal microbiota by increasing the number of beneficial lactic acid bacteria, namely, Lactobacillus, and reducing Gammaproteobacteria, which include several potential pathogenic bacteria. The specific composition of 1-monoglycerides of short and medium-chain fatty acids contained in SILOhealth 108Z could thus have a great potential as a feed additive in aquaculture.