Modulation in fish gut health transcriptome as a consequence of sodium butyrate supplementation

AQUAFEED

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Modulation in fish gut health transcriptome as a consequence of

sodium butyrate supplementation

Alvaro Ortiz, Aquaculture Product Manager, Norel Aquaculture is the world´s fastest food production system. Data from World Bank's Global Program on Fisheries and Aquaculture reveals that aquaculture must grow 75 to 85 percent by 2030 to supply the world's increasing demand for seafood (Anderson, 2011).

In order to achieve such development, nowadays, aquaculture is facing a number of challenges.

In first place, high stocking densities and the consequent proneness to pathogen transmission, water contamination and sediment deterioration.

In second place, the limited - or even banned - use of antibiotics and disinfectants, broadly used to control pathogens but lately under the spotlight due to the evidence that their use in animal production can lead to the natural emergence of bacterial resistances.

In both industrialized and developing countries, nutrition and feeding, play a critical role in the sustained development of aquaculture, thus particular emphasis is currently being placed on nutritional strategies and their effect on animal health, performance, and environmental integrity. The use of the increasingly accessible information on nutrient requirements for each of the species, as well as the use of alternative ingredients is the first approach. A second strategy is the supplementation with natural growth promoters (NGPs). Also termed as non-antibiotic growth promoters, NGPs are feed additives contemplating different categories as salts of organic acids, probiotics, prebiotics, phytogenics or yeasts. In addition to the beneficial effects in terms of health status and performance,

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NGPs supplementation does not usually bear any risk regarding bacterial resistance or undesired residues in the edible fish product.

Nutritionists must focus not only on nutritional specifications but also on promoting digestive health. In such task, probiotics and organic acid salts, two of the most well documented NGPs, have aroused as natural solutions that result in improved health status, nutrient utilization, and consequently performance.

Several publications have addressed the effects of short change fatty acids (SCFA) and their salts on the health and performance of farm animals. These SCFA are commonly known by their specific antimicrobial activity, however the effects of SCFA go beyond modification of gut microflora, and other benefits such as improved digestive enzyme activity, increased pancreatic secretion, enhanced development of intestinal epithelium and intestinal barrier integrity, or anti-inflammatory properties, have been described and attributed to likely causes of enhanced performance, particularly when supplementing butyric acid in its sodium salt form.

Butyric acid is naturally produced and present within the intestine of both carnivorous and herbivorous fish (Holben et al. 2002; Mountfort 2002) but, unfortunately most of the studies about butyrate capacities are conducted with land animals so the references for fish are scarce and mostly concentrated in salmonids.

In order to asses all these benefits in fish different from salmonids, a study was conducted by Jaume Pérez-Sánchez and his team at the “Instituto de Acuicultura de Torre la Sal”, IATS-CSIC and the University of life Sciences (Norway) in which the commercial butyrate product (BP-70, ®Norel) was tested on gilthead sea bream, a carnivorous fish of the bream family Sparidae adapted to warm water.

Juvenile fish of 25 g initial body weight were randomly distributed into 90-L tanks in triplicate groups of 15 fish each fed (9 weeks) with varying concentrations of BP-70, added to a plant protein-based diet with 20% of fish meal inclusion and 35% of fish oil replacement by a blend of vegetable oils. The diet designed was focused on the study of the effect of sodium butyrate when part of the marine raw materials are substituted by plant proteins and fats.

Focusing on intestine health parameters, gut index (fish weight/intestine length) was progressively and significantly increased with butyrate supplementation. Normally fish react to poor nutrient absorption enlarging the intestine increasing therefore the absorptive surface, this compact intestine could be indicative of better conformed microvilli. Butyrate also increased plasma glucose levels and liver glycogen depots, which highly supports a sparing effect of butyrate on the utilization of glucose as a metabolic fuel. This has also been reported in other species (Bergman, 1990; Guilloteau, 2010)

Stomach E-cadherin

Figure 1: Immunostaining of gilhead sea bream intestine with E-cadherin antibodies

Apart from the typical parameters, a transcriptomic analysis was conducted too. Techniques such as RT-PCR allow us to describe modifications in the expression of determined genes.

The amount of gene transcripts (messenger RNA: mRNA) can be robustly detected and later on analysed by computer software to calculate relative gene expression. This way, the effect of an addi-

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AQUAFEED

tive on gene transcription modulation can be objectively measured.

A gene expression array of 90 specific genes was used to characterize the intestine gene expression pattern. Butyrate supplementation clearly induced modifications towards a healthier intestine condition. Genes related to regulatory networks promoting intestinal cell differentiation rather than stem cell proliferation were found to be upregulated by the presence of sodium butyrate. This agrees with the lowered expression of the proliferating cell nuclear antigen (PCNA) at mRNA and protein level, as evidenced by RT-PCR and immunocytochemistry, respectively. So differentiation and thus specialization of enterocytes was promoted instead of proliferation which sometimes is related to tissue damage.

Butyrate supplementation also improved the intestine barrier function, up-regulating the expression of several components of tight junctions, as occluding, claudins and, tight junction protein ZO-1. Tight junctions proteins expression is crucial in terms of gut health.

They modulate permeability and prevent intracellular and intercellular infections as those caused by mixosporean parasites such as Enteromyxum leei.

At the same time, butyrate treatment altered the expression of nuclear-encoded mitochondrial genes, up regulating the expression of master transcription factors, mitochondrial protein translocases and oxidative enzymes, in addition to a down-regulated expression of mitochondrial molecular chaperones, which is prone to have a “high power mitochondria� phenotype with a low risk of cellular oxidative stress.

Figure 2: Summary of the transcriptome results.

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In addition, butyrate supplementation altered the expression of interleukin 7 (IL-7), nucleotide-binding protein oligomerization domain-containing protein 1 (NOD1), vimentin (VIM), macrophage mannose receptor 1 (MRC1) and C-C motif chemokine 20 (CCL20), leading to an anti-inflammatory gene expression pattern.

This study was presented in the Aquaculture conference: “To the Next 40 Years of Sustainable Global Aquaculture” that took place in Las Palmas during the 3-7 of November, 2013.

Looking at the modulations in the expression of these gut-genes, we can observe that all the modifications were oriented to an improved gut health status. Balance in the transcription of genes is essential for a healthy intestine: presence of enough functional enterocytes, epithelial integrity, powerful mitochondria and a correct immunomodulation are needed to have a correct intestine , this subsequently will enable the fully expression of the fish’s growth potential. Therefore, sodium butyrate appears to be a useful tool in the more and more difficult work of producing fish within the frame of sustainable aquaculture practices.

More information Alvaro Ortiz, Aquaculture Product Manager, Norel