By Milena Saqui-Salces, University of Minnesota Department of Animal Science assistant professor, Integrated Animal Systems Biology Team
Gut health is difficult to define because it is a concept that involves numerous aspects of the gastrointestinal tract function that promotes or results in animal well-being.
I discourage the use of the term “gut health” because I find many of the measurements reported in the scientific and commercial literature are inadequate to support the conclusions of GI function improvement in response to feed additives and nutritional interventions. Many studies overstate their findings by assuming physiological benefits from the changes observed, often failing to demonstrate improved function. This results in conflicting studies and widespread confusion in the pork industry when interventions that claimed to provide benefits to the pig do not show their effects on the farm.
With our current knowledge, the best we can aim for is to identify interventions that improve animal performance in specific conditions, and accept that there is not, and probably will not be, a single product that will work on all pigs, under all conditions.
The purpose of this article is to help pork producers, nutritionists and veterinarians understand the common gut health measurements reported in the literature and how to interpret their relevance for achieving improved performance in pigs. Understanding the “what” and how function is measured is very important to identify when determining if a claim of “gut health” promotion is supported or not.
The GI tract has four basic functions: digestion, absorption, motility and secretion. The gut also has a significant role in immune response because of its direct exposure to everything an animal consumes, including pathogens, antinutritional factors and toxins, as well as nutrients and beneficial bacteria.
The majority of studies reported in scientific literature involving pigs assess gut health status by analyzing some of the characteristics of the GI tract, which include maintenance of barrier function, intestinal morphology, immune response and the changes in microbiome composition. These properties affect the function of the GI tract and the whole animal, but none — on their own or in combination — guarantees improved pig performance. Defining the physiological characteristics and parameters of a “healthy gut” is difficult, but identifying an unhealthy intestine is relatively easy.
Most scientists evaluate either loss of function or the absence of disease as a measurement of gut health. The obvious indicator of disease or inadequate intestinal function is diarrhea. However, when attempting to improve animal performance, we need to focus on measuring indicators of subclinical conditions that compromise optimal growth.
In general, there are three ways to investigate GI function: in vivo, ex vivo and in vitro. Everything measured in a living animal, or from a sample that comes from a living animal, is considered to be an in vivo response. Ex vivo are all those measurements conducted using a sample obtained from a living animal but tested in the laboratory. When doing in vivo studies, sometimes animals are kept alive, but often they need to be euthanized to obtain the samples. Ex vivo studies almost always require the animals to be euthanized.
In vitro studies do not use tissues or samples obtained from animals, but use cells grown in the laboratory. In vitro studies are not adequate to prove function, but are a way to identify mechanisms of action and evaluate potential interventions.
From all the in vitro models, enteroids best represent the structure, function and dynamics of the intestine. Enteroids are derived from stem cells of the pig’s intestine grown in special culture medium. They can be used to evaluate the physiological effects of various compounds, and even to model intestinal diseases (see article on Page 12), without the complexity, time and expense of feeding these compounds to animals.
In vivo, the function of the GI tract can be evaluated with digestibility studies, evaluation of intestinal permeability, mucosal structure if animals are euthanized and systemic immune responses.
The most common example of ex vivo studies is the use of Ussing chambers. These chambers are devices with two sides connected by a small opening where a piece of intestinal mucosa collected from an animal immediately after euthanasia is securely sealed on both sides. As the only way that compounds can move from one side to the other is through this piece of intestine, Ussing chambers allow measuring the capacity of the intestine to transport nutrients, and determining whether the intestine is leaky or not.
“Leaky gut” is a term often used to describe a loss of intestinal barrier function or increased intestinal permeability. Intestinal permeability is the capacity of the intestine to regulate the transport of compounds that enter and leave the body. Two major conditions compromise the intestinal barrier: loss of tight junctions between epithelial cells (permeability) or damage to the epithelium (major structure changes) that results in partial or complete loss of the barrier.
Changes associated with tight junctions are probably the most commonly used to make claims about gut health in the literature. However, as we will discuss, unless the function is tested, most measurements involving tight junctions in the literature are uninformative of gut health.
Tight junctions are the structures that keep the intestinal epithelial cells together and block the passage of many substances into the body, while allowing the transfer of others. Tight junctions are comprised of different proteins that work together: claudins and occludins that are located in the junction, and zona occludens proteins that anchor claudins and occludins to the cell.
ZO proteins are not exclusive to tight junctions; they also form adherens junctions that help keep the cells together but have only minor roles regulating transport of nutrients and other compounds. Because of these reasons, analyzing ZO-1 or ZO-2, although commonly done in many scientific studies, is not informative for assessing the status of tight junctions. Instead, studies should focus on analyzing maintenance of the function (with Ussing chambers) or on location of claudins and occludins when evaluating tight junctions.
That said, reports of changes in gene expression of tight junction proteins or protein quantification in the tissue do not provide evidence of changes in tight junctions or intestinal permeability.
Currently, the gold standard for assessing intestinal permeability is the use of Ussing chambers to measure trans-epithelial resistance and permeability. TER is the measure of the electric resistance to current (ion flow) of the tissue. An intestine that has strong tight junctions will have higher TER than a leaky intestine.
Another approach used to measure intestinal permeability includes feeding molecules that would normally not be absorbed in the intestine (e.g. lactulose; polyethylene glycol; labeled dextran; mannitol; or a combination of lactulose, cellobiose, mannitol and L-ramnose) to measure their concentrations in urine or blood as indicators of increased permeability.
Despite the possible confounding factors of bacterial processing, and liver and kidney function, this approach is the best tool available at this time to assess intestinal permeability in vivo. Some researchers measure circulating bacterial endotoxins, endotoxin core antibodies (anti-lipid A antibodies) and D-lactate. The presence of these compounds in the blood is considered an indicator of loss of barrier function, but none is specific to the intestine, and can result from loss of barrier function in the respiratory or reproductive tract, skin, or other organs.
Another common assessment of GI tract health is a histological evaluation of mucosal structure. Mucosal structure refers to the general organization of the intestinal tissue, including the length of the villi, depth of the crypt, number of villi-crypt units by unit of length of the intestine, the presence of inflammation, and overall weight and size of the organs. Mucosal structure is highly informative when major changes are observed; for example, the villi in pigs infected with pathogens are usually blunted and half or less than half the length of the villi of a healthy pig, plus they show cellular damage.
However, when changes are not that drastic, like under stress or as a result of dietary interventions, interpreting changes of the mucosa becomes a challenge.
In the animal science field, it is widely accepted that longer villi are associated with greater absorptive capacity, and thus are interpreted as an improvement in gut health. However, not a single published study shows that intestines with longer villi indeed absorb more nutrients. Moreover, greater intestinal villi height and deeper crypts mean the GI tract may be heavier, which is undesirable relative to pig growth performance.
Several studies have shown increases in intestinal villi and crypt lengths in pigs — a result of feeding low-nutrient-dense diets. Authors suggest that is a normal response of the intestine to increase absorptive capacity to better use those nutrients. Following that thought, it would be even more useful to demonstrate more villi per length of intestine, which implies even larger absorptive capacity even if villi do not change in height; but this measurement has rarely been reported in scientific literature.
Contradicting the idea of longer villi happening in response to low nutrient density, it has been widely reported in many studies with nursery pigs and other animals, including humans and rodents, that under conditions of severe nutritional restriction and fasting, the absence of food in the intestine or low nutritional content results in villi becoming shorter, not longer. Therefore, interpretation of changes in villi height and crypt depth needs to be supported by functional measurements.
Currently, the optimal villus height and crypt depth, and the ideal proportion of these measures that should be in each section of a pig’s intestine at different ages to support optimal growth, are unknown. Overall, unless there is clear damage to the tissue or the villi are severely blunted, changes of intestinal morphology do not provide evidence of gut health.
The intestinal barrier is essential for optimal immune function and health of pigs. Immune response is measured by quantifying the concentrations of cytokines, interleukins and other molecules in the blood by ELISA, as well as identifying the immune cells present in the tissue or blood (usually by flow cytometry.) Both cells and molecules can be measured in vivo, ex vivo or in vitro.
In general, in vitro and ex vivo studies are meant to understand mechanisms of immune response and do not accurately model responses in vivo. In vivo studies are conducted to evaluate immune responses triggered by nutritional interventions or under-stress challenges (heat or social) to determine if the immune system is being activated even without clinical manifestations.
Other studies are conducted using a stress or disease challenge to measure the efficiency and ability of animals to respond, control and recover from a challenge.
When evaluating immune responses associated with the GI tract, blood samples collected from the portal vein or intestinal tissue are necessary because changes in the local intestinal immune response may not be observed in systemic blood (samples collected from the jugular, ear veins or other than the portal vein). This is because some cytokines and immune factors may be cleared by the liver, and the systemic blood carries signals from the respiratory and reproductive tracts, skin, and other organs, in addition to the intestinal tract.
Also, comparing concentrations of cytokines from swine in different production phases (gestation, lactation, nursery, finishers) should be avoided because the concentrations vary by age. In pigs, several scientific studies have provided information on pro-inflammatory molecules, particularly IL-1β and TNFα, as well as antibody production using different models, but the information about the quantification of the impact of immune response on performance is not clear when challenges are not included in the experimental design. The lack of significant changes may be a result of a limited focus by analyzing only pro-inflammatory cytokines and overlooking other components of the immune response, such as the anti-inflammatory, regulatory and memory that are rarely studied in research involving food-producing animals.
The microbiome is unquestionably one of the more important factors affecting GI tract function, intestinal barrier and immune responses. Unfortunately, researchers are just beginning to identify changes of the gut microbiome resulting from various environmental and nutritional conditions, and to understand what the changes mean relative to pig performance and health. Most of the studies involving the modulation of the pig microbiome involve large, complex data sets generated from sequencing bacteria from feces or culture to identify different microbial species or families.
At this point, the best microbial composition for the pig’s intestine has not been defined, and most statements that refer to good or bad bacteria are based on information from human or mouse studies. However, research is beginning to show how differences in microbial composition of the GI tract may be correlated with a pig’s health and performance. Eventually, the goal is to evaluate and validate cause-and-effect relationships between these correlations.
Remember, the microbiome is comprised of not only bacteria, but also fungi, protozoans and viruses. Unfortunately, there is little scientific information on the role of these nonbacterial components of the microbiome in GI function and interaction with other components.
In summary, the functions of the GI tract of pigs are complex, and their study requires using different research techniques and measurements. At a minimum, these measurements should be tested for correlation with animal performance to provide meaningful interpretations of their significance for pig’s health.
Furthermore, since improving animal performance involves all the systems in the body, a holistic approach needs to be used to identify genetic, phenotypic and metabolic biomarkers that identify health in pigs to be used as predictors of performance in commercial production, considering that gut health may be only one small part of the puzzle.