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Confirming gut health without touching the pig

Intestinal integrity refers to how well the structure of the epithelia is maintained. It is increasingly possible to establish gut integrity in live pigs without using invasive methods. Applying this method, it was possible to establish the effects of a β-mannanase enzyme on gut integrity in weaner pigs.

The intestine contains epithelial, immune and nervous tissue that regulates the integrity of the epithelial barrier as well as the different functions of the digestive system (degradation of feed and ­absorption of nutrients).

Weaning starts a critical period in the life of piglets, where the integrity of the intestinal barrier is often damaged, which typically leads to gastrointestinal disorders that can be aggravated by different pathogens and antinutritional factors and further disrupt the intestinal epithelia and its ability to absorb nutrients. These morphological and functional changes can have long-term consequences that reduce both animal health and productive performance.

Tight junctions are a critical component of the intestinal epithelia, because they regulate the space between epithelial cells to allow certain nutrients to pass, while micro-organisms and other intestinal content are blocked. Intestinal integrity refers to how well the structure of the epithelia is maintained. A healthy or intact epithelia generally means that the epithelial cells are firmly attached to each other, that nutrients are efficiently absorbed and that its critical function as a barrier against infections is effective.

This article discusses the use of a non-invasive monitoring system based on analysis for intestinal biomarkers in faeces samples collected from average, apparently healthy pigs to gain information about intestinal integrity and the level of immune activity/inflammation. Results from a nursery trial with β-mannanase will be discussed, where the system was used to assess the effects on intestinal integrity and ­inflammation.

Trials in piglets weaned at 21 days of age showed that adding a β-mannanase enzyme led to better weight gain and feed efficiency. Photo: Bert Jansen
Trials in piglets weaned at 21 days of age showed that adding a β-mannanase enzyme led to better weight gain and feed efficiency. Photo: Bert Jansen

How to assess the intestinal integrity status

Different approaches can be used to investigate intestinal integrity, and there is still much to learn about the best way to use them. The chosen method here is to focus on gene expression analysis that measures the amount of mRNA coding for different markers associated with intestinal health, because the technique is well established and relatively economical.

Although many different markers can be considered, markers were selected that can be evaluated via rectal faeces samples. Specifically, analysis was conducted for mRNA expression for occludin, zonulin, interferon-gamma (IFN-γ), Transforming Growth Factor beta (TGF-β) and calprotectin (Figure 1).

  • Occludin and zonulin are tight junction proteins that, simplistically, can be considered part of the glue that keeps the cells of the intestinal epithelia connected. Reduced expression of occludin and zonulin in faecal samples therefore indicates reduced intestinal integrity, inflammation and ­increased permeability.
  • IFN-γ is an important proinflammatory cytokine that also plays a regulatory role in the immune response and ­inflammation.
  • TGF-β is a multifunctional cytokine that plays an important role in regulating the inflammatory process that is often considered anti-inflammatory, although it can also have ­proinflammatory properties.
  • Calprotectin is a marker for intestinal inflammation because elevated values in faeces indicate that white blood cells (neutrophils) have leaked into the luminal content.

Zonulin and occludin are indicators for the status of tight junctions and thus intestinal integrity. Calprotectin, IFN-γ and TGF-β are indicators of immune activation and the inflammatory process, and the levels of calprotectin and IFN-γ are expected to decrease when intestinal inflammation is reduced, while the levels of TGF-β, zonulin and occludin can be more difficult to interpret.

Figure 1 – Expression of markers for intestinal health.

Confirming gut health without touching the pig

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Biomarkers used to assess gut integrity

Hemicell HT (Elanco) is a β-mannanase enzyme that degrades highly antinutritive β-mannan fibres found in most vegetable feed ingredients. The content in most of them is low, well below 1%, so typical swine diets are unlikely to contain more than 0.2–0.4% soluble β-mannan. However, their presence in common diets has been associated with a broad range of adverse effects. Mostly, these can be linked to the ability of β-mannan to provoke a wasteful immune response that causes intestinal inflammation and leakage that may increase ­susceptibility to disease challenges.

The tool was used to evaluate intestinal health in the second batch of a recent nursery trial with piglets weaned at 21 days of age, conducted in France by MG2Mix (Table 1). Standard control diets were compared to diets with 300g/tonne of the β-mannanase enzyme, where 2% soy protein concentrate was replaced with 2.7% soybean meal in phase 1, 6–10kg, and where the enzyme was formulated into the feed to replace 63kcal/kg NE in phase 2, 10–25kg. In total, 15 faecal samples were collected from each treatment at the end of the second batch, 41 days post-weaning.

The piglets receiving the β-mannanase enzyme had better weight gain and feed efficiency and higher feed intake than the control pigs. Equally importantly, no differences were observed in health, faecal scores or mortality and no antibiotic treatment was needed during the trial. Knowing that improved intestinal integrity is typically accompanied by higher performance it was interesting to observe the results of the analysis for the five markers.

Level of immune activity

That the expression of calprotectin and IFN-γ were significantly lower (p<0.05) on hemicell ht compared to the control group indicates that the levels of immune activity and intestinal inflammation were reduced with the enzyme, which was expected because it reduces the ability of β-mannan to provoke an immune response.>

That the expressions of TGF-β and the two tight junction proteins, occludin and zonulin, were similar on both treatments may be more difficult to interpret. However, the good health status of the pigs likely played a role, so the results from the tool seem to support the performance differences.

There is still much to learn about how to interpret the results, and the tool itself can likely also be improved, but the preliminary results are very promising because they indicate the tool may be useful to evaluate intestinal health in piglets.

References available on request

Authors: Dr Pedro Jose Sanchez and Karl Poulsen, Elanco Animal Health and Professor Guillermo Ramis Vidal, University of Murcia, Spain