Numbers of feed additives have more than one function and some of them are now under study for their possible positive effect on animal health and performance. Health is the starting point to improve production and intestinal health is the basis for general health. The effect of a feed additive can be measured not only from a nutritional standpoint but also by how it can sustain animal physiology and general/intestinal health. The impact that a molecule can have on animal metabolism and physiological functions is strictly linked to different modes of action of the molecule itself. These activities must be studied and well known to use the nutrition favoring both animal health and production effectively.
Organic acids: not only preservatives
Organic acids (OA) have been commonly used for thousands of years as food preservatives in animal nutrition due to their broad-spectrum antimicrobial activity. They can contribute to feed hygiene by reducing the growth of bacterial pathogens and molds, therefore allowing better use of feed resources (Khan & Iqbal, 2016). The anti-microbial activity can vary among different OA and it has been shown to be dependent on many properties of the molecule itself (Hsiao & Siebert, 1999). In general, considering the varying sensitivity of the different classes of microorganisms toward different organic acids, blends of OA and their salts are effective against a wider range of microbes than single acids.
Organic acids are also successfully used in animal production as an alternative to antibiotic growth promoters to improve animal live performances (Khan & Iqbal, 2016). This kind of use is linked not only to antimicrobial activity but also to the involvement of some OA in specific metabolic pathways. As an example, sorbic acid is a poly-unsaturated fatty acid (PUFA) with a conjugated double bond, discovered to regulate IGF system gene expression and hormone secretion, stimulating animal growth (Luo, et al., 2011).
Pure botanicals: different molecules, numerous benefits
Botanicals are the most studied alternatives to antibiotic growth promoters of the last ten years. Plenty of literature reports the use of botanicals in the animal feed industry and they generally refer to them with a lot of different names like plant extracts, phytogenics, essential oils, and so on. The term “essential oil (EO)” refers to the volatile lipophilic constituents extracted by several parts of plants (flowers, seeds, leaves, roots, etc., …), so this extract is a blend of various compounds (Amorati, et al., 2013). According to the plant chosen, one or more compounds are dominant but there are numerous different active compounds and the percentages found are variable. The content of active substances within a certain oil may vary widely, depending on the plant part used, harvesting season, and geographical origin. Some isolates are fractionally distilled and could have a small amount of impurities associated with them (Windisch, et al., 2008; Furia, 1980). Pure botanicals (PB) are chemically defined substances that constitute a plant EO, so they are produced synthetically but are chemically identical to their natural counterparts. Pure botanicals include many classes of molecules, most of which have proved antimicrobial (including antibacterial, antiviral, and antiprotozoal) and immunomodulatory activity, connected with their antioxidant and anti-inflammatory action. The mode of action in this way is different for each molecule class: i.e. polyphenols scavenge free radicals, terpenes interact with metabolic pathways and enzymes, etc. (Brenes & Roura, 2010).
Intestinal health: the key for healthy and productive animals
There are more and more pieces of evidence about the importance of gut health for general health. The gastrointestinal tract is the first contact of the organism with the environment with the ingested diet both as an essential source of nutrients and a harmful vehicle for pathogens. The intestinal mucosa has the double role of absorption of useful molecules and barrier against pathogens and toxins. Its epithelium is composed of a single layer of cells connected to each other through inter-epithelial protein structures, the so-called thigh junctions (Pitman & Blumberg, 2000). The maintenance of the mucosal barrier function contributes to the homeostasis and health of the animals, while its impairment may lead to an increase in clinical pathologies and the reduction of animal growth performance (Moeser, et al., 2007). Thigh junctions are composed of more than 30 proteins (zonula occludens and occludin are among the most important ones to maintain the structural function): a dysregulation of thigh junctions enables harmful substances to translocate, resulting in damaged tissue and inflammation (Anderson & Van Itallie, 2009; Tsukita, et al., 2001).
The impairment of the intestinal mucosa barrier function occurs in case of pathologies but also in different stressful periods, unavoidable during the rearing cycle, such as the hot season and heat stress, weaning, regrouping, and so on (Campbell, et al., 2013; Fontoura, et al., 2022). Strategies to sustain the intestinal mucosa physiology and function are needed, to support and improve animal health and, consequently, their performance.
How OA and PB can help?
OA and PB have both antimicrobial properties: their different modes of action allow a synergistic effect against pathogens. Anyway, their synergy is not only linked to antimicrobial activity. These molecules have numerous biological properties and can exert a role in the maintenance of intestinal functionality (Tugnoli, et al., 2020; Rossi, et al., 2020; Khan & Iqbal, 2016). In particular, the beneficial effects of a blend of OA (citric and sorbic acid) and PB (thymol and vanillin) on porcine intestinal barrier function, through the stimulation of the local immune response, the increase in the mucosal transepithelial electrical resistance (TEER), and the reduction of the intestinal permeability to dextran (paracellular permeability, PCP), were reported (Grilli, et al., 2015).
Recently Vetagro conducted, in collaboration with the University of Bologna (Italy), a study aimed to explain the mechanism of action of the active ingredients of AviPlus® (citric and sorbic acid, thymol, and vanillin, OA and PB contained in this Vetagro product) in preventing or improving intestinal barrier failure (Toschi, et al., 2020). The study was conducted in vitro to exclude any interference due to the intestinal microflora of the animals. Molecules were tested on Caco-2 cells, an internationally recognized model for the intestinal epithelium.
First of all, OA and PB were tested alone and in combination in a dose-response test. Results indicated a positive and dose-depending effect on the intestinal barrier function for the single molecules but especially for their combination. The experimentation demonstrated that the epithelia treated with OA, PB or their combination (Figure 1) had higher TEER compared to the control.
Figure 1: TEER of Caco-2 cells cultured with the mix of OA and PB included in AviPlus® (citric and sorbic acid, thymol and vanillin). Data in the graph are represented as a percentage over the initial TEER value and given as means (n = 6) ± SEM, represented by vertical bars. Means with different letters (a, b) indicate statistical significance with P < 0.05, while means with at least one common letter are not statistically different. Treatments were: control (CTR), no treatment; OA+PB 200, treated group with 200 ppm of the mix; OA+PB 1000, treated group with 1000 ppm of the mix.
The second part of the study aimed to investigate the effect of the combination of the OA and PB of AviPlus® in improving the intestinal epithelium reaction against an inflammatory challenge (experimentally induced by using bacterial LPS and pro-inflammatory cytokines). In particular, two experiments were set up, both lasting 15 days: in the first one the inflammatory challenge was done on day 0, while in the second on day 14. Results showed that OA and PB synergistically improved the effectiveness of the barrier function in both experiments. In particular, when the challenge was at the beginning of the test (Figure 2-a), the TEER of the treated epithelia increased significantly more than the control and remained higher throughout the whole trial. Also in the second test (Figure 2-b) the inclusion of the two dosages of the mix led to an improvement of the TEER before the challenge on day 14. On the last day of the experiment, all groups had a reduction in the TEER due to the challenge but, notably, the higher dosage of the combined molecules allows cells to restore the initial TEER within 24 h as if there had been neither the treatment nor the challenge. The same and dose-dependent effect was recorded in the gene expression of zonula occludens and occludin in both tests, indicating an improvement in the tightness of the cellular monolayer due to the treatment with OA and PB.
Figure 2: TEER of Caco-2 cells cultured with the mix of OA and PB included in AviPlus® (citric and sorbic acid, thymol and vanillin). Data in the graph are represented as a percentage over the initial TEER value and given as means (n = 6) ± SEM, represented by vertical bars. Means with different letters (a, b) indicate statistical significance with P < 0.05, while means with at least one common letter are not statistically different. Treatments were: control (CTR), no treatment; OA+PB 200, treated group with 200 ppm of the mix; OA+PB 1000, treated group with 1000 ppm of the mix. a) Inflammatory challenge on day 0; b) Inflammatory challenge on day 14.
Conclusions
A healthy gut is the basis of animal health and productive performance. Since the intestine is exposed to different pathogens and harmful substances, it is essential to maintain the mucosa barrier function to prevent health problems. Nutrition is among the principal ways to sustain gut health. Organic acids and pure botanicals are widely used in animal nutrition: it has now been shown that their synergistic action leads to significant improvements in intestinal morphology and barrier function, both in physiological conditions and in case of challenges. For more information: marketing@vetagro.com