Antimicrobial growth promoters have attributed to the prevention of gastro-intestinal bacterial infections instigated by various stress factors. The economic importance of gut health issues in farm productivity is well established and resolving these issues is a main point of focus. Next to this, consumer, and hence also governmental, concern has never been more focused on food safety and traceability. For example, the European Union (EU) has set clear targets to combat Salmonella and strict EU regulations have been implemented, bringing an added cost to animal production for control and monitoring programmes.

Modern production animals are mostly fed on easily degradable diets, meaning the ‘feed processing’ is done already in the manufacturing plant. As a consequence, an adequate development of the gut is hampered and feed passes very quickly through the intestinal tract allowing for a too short time for complete digestion. This is especially a problem for protein digestibility. Poor protein utilisation results in an excess of undigested protein in the intestinal tract which then increases the risk for bacterial imbalances. Up to now, these imbalances were often corrected by the use of antimicrobials.

In the near future, high-quality ingredients (e.g. high-quality protein sources) will become even less available for animals as human consumption will get priority. Animals that are being fed lower quality ingredients have an increased risk for health issues promoting the need for preventive and/or curative treatments.

With the recent and projected increase in the human population as well as the rise in income and as a consequence a rise in demand for animal products worldwide, the identification of novel strategies to increase the efficiency of food production has become a major focus. With respect to agricultural animals, optimisation of animal health and nutrient utilisation are key components for improving production efficiency, a responsible use of antimicrobials and increasing food safety. The need for a highly effective prophylactic approach to animal health is clear.

In recent years, a number of different molecules and additives have been tested on their ability to improve gut integrity. Classes of additives include amongst others prebiotics, probiotics, botanical products, acids (inorganic, organic, short and medium chain fatty acids) and enzymes.

In order to evaluate these compounds, one should aim at understanding as much as possible about their mode of action. The intestine is a complex system in which several factors influence the final outcome. In the intestine, three major components (the mucosal barrier, the composition of the microbiota and the local immune system) provide defensive measures against different pathogens through permanent contact and dialogue with each other. Feed additives may interact with host cells (intestinal cells, immune cells), with the host microbiota or with pathogens impairing the normal intestinal function. A major concern in the development of new strategies is the lack of knowledge of the mechanisms through which the substances work.

Butyrate: Mode of Action

The short chain fatty acids (SCFA) constitute a group of molecules that contain from one to seven carbon atoms and which exist as straight or branched-chain compounds: predominant SCFAs are acetic, propionic and butyric acids. Because SCFA are weak acids with a pK of 4.8 and the pH of the gastrointestinal tract (GIT) is nearly neutral, 90 to 99 per cent of the SCFA are present in the GIT as anions rather than free acids.

Among the SCFA, butyric acid has received particular attention. Butyric acid is available as the sodium, potassium, magnesium or calcium salts.. The advantage of salts over free acids is that they are generally odourless and easier to handle in the feed manufacturing process owing to their solid and less volatile form. For the purpose of the present article, the term ‘butyrate’ is used interchangeably for the acid, the salt and the anion forms.

Recent research carried out in human health indicates some interesting results. In humans, the effects of butyrate can be subdivided into intestinal and extra intestinal. Intestinal effects include the following: butyrate has a double effect on cell growth, defined as the “butyrate paradox”; it stimulates physiological proliferation of normal enterocytes, whereas it inhibits cell proliferation in a colon carcinoma cells (Scheppach & Weiler, 2004) providing possibilities for preventing and inhibiting colon carcinoma.

Butyrate has also been shown to have anti-inflammatory effects as well as antioxidant activity (Sossai, 2012). This anti-inflammatory action of butyrate has been the subject of some interesting studies regarding its use in inflammatory bowel diseases (Crohn’s disease).

Another interesting application of butyrate involves its use in irritable bowel syndrome (spastic colon), of which knowledge is still fragmentary, especially regarding the enteric nervous system and its regulation (Sossai, 2012). Extra intestinal effects are less well known but equally intriguing: butyrate shows promising results for treatments of hematological diseases (e.g. thalassemia; sickle cell anemia), hypercholesterolemia (butyrate can down-regulate the expression of genes involved in the biosynthesis of cholesterol and triglycerides), reducing resistance to insulin (in animal studies), and reducing vascular stroke (in animal studies) (Sossai, 2012).

Intriguing results such as described above plus research done before have stimulated further in-depth investigations both in humans and in animals to learn more on the mode of action of butyrate. In short, butyrate may function as a ligand for transmembrane receptors, as a modulator of gene activity, and as a direct energy source for cellular metabolism. Several cell types, many of which associated with the digestive tract, have been described to be receptive to one or more of these biological effector functions, which explains the extensive range of biochemical effects that have been documented to be mediated by butyrate.

Butyrate, when present in the blood stream or in the proximal parts of the intestinal tract, induces the production of host defense peptides (Guilloteau et al., 2009). These peptides stimulate the development and repair of the intestinal tract through an increase in cell proliferation (Bartholome et al., 2004). Recently it has been shown that butyrate, when present in blood, stimulates a peptide that increases the absorption of glucose from the intestine. Indications that a similar mode of action can be expected in poultry, is shown by Hu and Guo (2007), who found an increased development of the villi when sodium-butyrate was added to the diet.

Butyrate also has been shown to stimulate several functions in the lower part of the intestinal tract. Studies have identified specific G-protein-coupled receptors, specifically GPR 41 and GPR 43, on gut epithelial cells in the epithelium of particularly the ileum, caeca and colon (Le Poul et al., 2003). When butyrate is attached to these receptors the production of several different peptides is stimulated (Cox et al., 2009, Tazoe et al., 2008). Some of these peptides have a positive effect on the development of the immune system and improve the functioning of the immune system in case of a health challenge (Cox et al., 2009). Other peptides have been shown to optimize gut motility, by delaying feed passage (Tazoe et al., 2008). The emptying of the feed out of the gizzard into the small intestine is slowed down. Thus, it seems that butyrate is inducing a similar effect to passage rate as coarse particles.

Indications that butyrate also stimulates the immune system in poultry were obtained by Leeson et al. (2005): birds previously fed butyrate showed a better withstand against the stress of coccidial challenge at 21 d of age. Weber (2008) found when pigs where challenged with Escherichia coli lipopolysaccharide (LPS), sodium butyrate increased the magnitude of the cortisol response and increased skeletal muscle IL-6 mRNA expression, also indicating that dietary butyrate affects the response to inflammatory stimuli.

In summary, beneficial effects include, among others, stimulation of digestive enzyme production, enhanced development of intestinal villi, reduction of inflammatory responses, increased immunity, reduced GIT retention time, inhibition of cancer cell growth and the secretion of host defense peptides (Guilloteau et al., 2012). Slower feed passage results in improved nutrient (protein) utilization. Improved immunity and a stronger intestinal barrier will result in a lower sensitivity of the animal to disease and hence may help to lower the need for therapeutic medications. This would have important positive implications for animals, farmers and society.

Apart from effects in eukaryotic host cells, butyrate is also described to have an impact on the activity of prokaryotes residing in the animal’s GIT. For example, it has been shown to affect the colonization of Salmonella and Campylobacter and to influence the composition of the gut microbiota (see below).

Butyrate: Experimental Evidence

Mucosal barrier

Dietary supplementation of butyrate has been shown to support enteric development and intestinal health of neonatal animals. At weaning, the small intestine of the piglet generally undergoes a decreased capacity of absorption that is associated with a marked reduction in villous height and crypt depth. These changes are accompanied with a decreased feed intake and poor growth (Piva et al., 2002; Pluske et al., 1996). Butyrate stimulates epithelial cell proliferation resulting in a larger absorptive surface, leading to improved feed utilisation. Furthermore, butyrate in the weaner diet preserves villus length and thereby helps to maintain feed intake. Effect of butyrate on gut morphology is of great biological value to the weaning period when the weight of the small and large intestine increases three times faster than that of the whole body mass.

The following trial results (North Carolina State University, Professor Ferket and colleagues, 2010) were obtained in broilers but show the principle of the impact of butyrate supplementation on intestinal development and growth performance in young animals.

Commercial broilers were randomly assigned to 32 floor pens containing 30 birds each and provided feed and water ad libitum until 49 days. Starter feed (pellet-crumbled) treatments consisting of 3 dietary supplementation levels of coated butyrate (0.0, 0.015, 0.03 and 0.06 per cent butyrate) were subjected to eight replicate pens per treatment from 1 to 14 days. Subsequently, all birds were fed common grower and finisher diets in pelleted form.

Bodyweight (BW) and feed intake was determined at 7, 14, 21, 42, and 49 days and feed/gain (FCR) calculated. At three, eight and 14 days, four birds per treatment were sampled for gut histology evaluation.

BW at 14 days increased linearly (p<0.01) as the level of butyrate increased (457g vs. 470g for 0 vs. 0.06 per cent butyrate) but no effects on 1-14 days FCR were observed. Histomorphometic analysis correlated with early treatment effects on BW (see Figures 1A and 1B).

The positive starter feed treatment effects was observed throughout the experiment, with 0.015 per cent butyrate resulting in a 3 per cent and 2 per cent improvement in 42 days (p<0.02) and 49 days (p<0.10), respectively. A linear improvement in 1-42 days FCR by up to three per cent was also observed as the level of butyrate increased in the starter feeds. Dietary supplementation of coated butyrate in starter feeds showed to have a lasting positive effect on broiler growth performance.

Figure 1a. Broiler chicks, 7 days, negative control

Figure 1B. Broiler chicks, 7 days, 0.06 per cent butyrate

Modulation of microbiota and impact on pathogens

Studies done by Galfi and coworkers (Galfi et al., 1991) have shown that butyrate increases the number of intestinal lactic acid and lactobacilli in butyrate-fed pigs, while decreasing the number of coliforms and E. coli. In his PhD-thesis, Pérez Gutiérrez (2010) investigated microbial composition after including different additives in the feed of (weaning) pigs. The qPCR-data revealed an increase in lactobacilli in the butyrate group, while weaning piglets that received butyrate also had a more homogeneous microbial profile, which was regarded as a positive effect.

In studies of Van Immerseel and co-workers, it was shown that butyrate, when present in the intestinal tract, was able to decrease Salmonella colonisation in broilers (Van Immerseel et al., 2005). The mode of action of this activity of butyrate seems at least partially mediated through modulation of gene expression. Butyrate specifically down-regulates Salmonella Pathogenicity Island 1 (SPI-1) gene expression, hereby preventing invasion of intestinal epithelial cells, one of the important steps of Salmonella pathogenesis in the bird (Gantois et al., 2006).

Van Immerseel and colleagues (2005) also demonstrated the importance of an effective coating in order to get significant reduction in Salmonella colonisation in the ceca and internal organs in vivo (Figure 2).

Figure 2. Percentage of animals with a certain amount of Salmonella in caecum (n=25 per group).

A trial done at the University of Bologna (Professor Bosi and colleagues, 2010) was done to look at the possible impact of butyrate (coated and uncoated) on E. coli K88 (ETEC) infection in piglets. Fifty-four piglets (prone to ETEC intestinal adhesion) weaned at 21 to 28 days were used (three control groups of six animals, three challenged groups of 12 animals, balanced for litter and weight).

Experimental diets were obtained with the addition of free or fat coated sodium butyrate. Full factorial design of two factors (four diets x challenge (E. coli K88) – yes/no), so groups were:

1. control diet, unchallenged
2. 2kg per tonne uncoated butyrate, unchallenged
3. 2kg per tonne coated butyrate, unchallenged
4. control diet, challenged
5. 2kg per tonne uncoated butyrate, challenged, and
6. 2kg per tonne coated butyrate, challenged.

The challenge with ETEC increased mortality in the control group (15 per cent mortality) and had an impact on the growth rate of the piglets. The piglets receiving the uncoated sodium butyrate showed a lower mortality rate (five per cent) and were to a lesser extent influenced by growth than the challenged control group. In the group offered the coated sodium butyrate in the diet, none of the piglets died and growth rate was only marginally influenced.

Butyrate: More Than Just the Active Compound

The effects on immune development, gut motility as well as the Salmonella effect via gene expression are only possible when the butyrate arrives in the lower parts of the intestinal tract. It is generally accepted that unprotected butyrate is quickly absorbed in the proximal part of the intestinal tract. Therefore, in order to get butyrate in significant levels available in the lower part of the intestinal tract the butyrate needs to be protected to achieve a target release.

In recent research done at the University of Illinois (Professor Hans Stein and colleagues, 2012) the importance of a good coating was again demonstrated. The researchers investigated the disappearance kinetics of different sources of butyrate in diets fed to weaner pigs.

Weanling pigs (n=24; 8.0 ± 0.5kg BW) were randomly allotted to three dietary treatments (six replicate pigs per dietary treatment):

1. a control diet
2. the control diet + 4kg per tonne of an uncoated butyrate (50 per cent product, UCB)
3. the control diet + 4kg per tonne of a coated butyrate (30 per cent product, CB).

The dietary treatments were provided to pigs daily for seven days as three times the estimated energy requirement for maintenance. On the last day of the experiment, all pigs were euthanized to collect samples of contents in the stomach, duodenum, jejunum, ileum, cecum, and proximal and distal colon. Concentrations of dry matter (DM) and butyrate were analyzed in all samples.

There was a similar pattern of the concentration of butyrate in the digestive tract, indicating that the concentration of butyrate was greater in the stomach than in the duodenum and jejunum, and gradually increased in the ileum. Weanling pigs fed the CB diet had greater (P<0.05) concentrations of butyrate in the jejunum (190 per cent g butyrate/g digesta DM) and ileum (188 per cent g butyrate/g digesta DM) than weanling pigs fed the control diet (µg butyrate/g digesta DM put as 100 per cent). In the caecum and colon, endogenous production was more variable among piglets and therefore no significant differences could be demonstrated.

In conclusion, the coated butyrate in spite of lower percentage of butyrate (30 per cent), increased significantly the concentrations of butyrate in the lower intestinal contents of pigs. Supplementation of the more concentrated (50 per cent) but less protected butyrate did not result in significant differences in the amount of butyrate in the intestinal tract compared with the control group.

Figure 3. Amount of butyrate in per cent compared to the unsupplemented control group.

Conclusion

Butyrate is a molecule of great interest to both human and veterinary medicine. Owing partly to the interest from human medicine, more and more profound knowledge on the mode of action has been obtained which allows more sound advice to producers.

In the current paper an attempt is made to summarise some of the new developments made on the research front as well as to show some trial results in production animals since in the end the benefits to the efficiency of animal production are what matter.

As shown above, butyrate has an effect on several levels (mucosal barrier, feed passage, microbiota, immune system, pathogens and others) and this combination of effects contribute to its general acceptance as helping for improved health as well as for improved performance.

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March 2015