There are numerous studies by our laboratory and others highlighting the benefit of using encapsulated ingredients for targeted release in the poultry GIT [10, 21, 27, 29-34]. In a previous study, the blend of organic acids and botanicals evaluated herein enhanced gut immune and barrier function in the ileum and jejunum of weaned pigs . In a separate study designed to begin to understand the mode-of-action at the gut level, a kinome analysis of ileal and jejunal samples collected from chickens revealed both common and distinct signaling pathways and proteins that were activated in each tissue compared to control-fed chickens . However, neither of the above-mentioned studies considered the role and impact on the gut microbial ecology; therefore, in the present study we determined the microbial populations of the ileum and jejunum from supplement-fed chickens compared to controls to provide additional insight.
Evidence suggests the stability of the microbiota is defined over time; however, changes observed in stable systems by compartment (tissue segment) can also indirectly support what is or is not viewed as a stable microbiota that may contribute to a loss in homeostasis [35, 36]. A classic example of the breakdown of gastrointestinal homeostasis is the emergence of ecological dysbiosis resulting in the de-compartmentalization of the gastrointestinal microbiota [19, 37]. The current study was not focused specifically on homeostasis or dysbiosis over time, but the findings herein indicate dietary supplementation with organic acids and natural compounds did result in significant compartmentalization of the microbial ecology within the ileum and jejunum of chickens. Each compartment functions independently with nutrient digestion and absorption typically occurring in the jejunum with water and mineral adsorption generally taking place in the ileum [38, 39]; therefore, it would be expected that the microbial populations would, in fact, differ between the two compartments. Additionally, while tissue differences in the microbial makeup exist comparing the NTC to tissue from supplement-fed chickens, there is not a collapse and shrinkage in diversity or a bloom of populations. These data are in agreement with other studies looking into nutrition and gastrointestinal health studies [35, 36]. While we cannot speak to potential changes or stability over time, data presented does indicate the microbiota is biologically diverse at 15 d-of-age in chicks provided a diet supplemented with the microencapsulated blend of organic acids and botanicals. However, some authors suggest the gut and microbiota at 15 d-of-age is only semi-developed ; therefore, future studies should consider the microbiota populations over the typical 42-day grow-out period.
Alpha diversity speaks to the community structure and evenness of the microbial ecosystem without taking into account differences in speciation while Shannon’s diversity index is classically associated with numerous microbial studies and is used to calculate evenness . Beta diversity indicates there may be compositional differences that are arising, with Bray-Curtis being a function of total assessment and the Weighted Unifrac Distance Matrix considering phylogenetic branch length and both are considered qualitative as total reads and counts leading to the differences are not considered . Dietary supplementation with natural compounds including organic acids and essential oils does not always result in changes to alpha and beta diversity in microbial populations within the poultry GIT . However, the blend of organic acids and botanicals used in the current study, produced an increase in diversity and evenness for the jejunum compared to the ileum. Similarly, in other pharmacological studies, the biotransformation of drugs by the microbiota results in their absorption in the jejunum and are linked to increased diversity and biological activity of the microbial population [41, 42]. The jejunum is the main sight for nutrient absorption in poultry , as well as in mammals, and it has been suggested that the jejunum is the most logical site to observe treatment effects  which is what we observed in the current study. Also, some feed additive studies utilize traditional culture-dependent microbiological evaluation to characterize the GIT microbial populations [28, 43, 44]. While these studies are valid and valuable, they are unable to take into account compositional and diversity changes. Therefore, the culture-independent study herein provides a deeper insight into the complete microbial shifts in two diverse and bioactive components of the GIT.
Natural compounds such as oregano and its derivatives, including thymol and carvacrol, are recognized for their potential benefits to the poultry industry because of antimicrobial properties and animal health benefits . Additionally, dietary supplementation with thymol has shown to increase Lactobacillus populations in the ileum [24, 43]; however, in the current study the changes in Lactobacillus populations were more pronounced in the jejunum compared to the ileum. This dissimilarity is likely attributed to experimental design differences including, but not limited to, the delivery method (non-encapsulated vs encapsulated), the genetic line of chickens used (Arbor Acre vs Cobb), the feed additive, or the thymol concentration (25% vs 1.7%). Even though the tissue-specific changes were different than the aforementioned study, our findings are in agreement with another study showing that inclusion of thymol does alter the GIT microflora of poultry . Another natural compound, a green tea component, also resulted in increased Lactobacillus in the jejunum compared to the ileum when fed to chickens . Collectively, these studies indicate an important role for the inclusion of thymol and other natural compounds into the diet as antibiotic alternatives.
In addition to increased Lactobacillus populations, other favorable changes were observed following supplementation including significant changes in Clostridiaceae in the 500g/MT jejunal samples. Similarly, supplementation with eugenol, an essential oil, increased members of the Clostridiales order in mice that proved protective against pathogenic challenge . There are a number of studies employing supplementation with natural products including organic acids and phytochemicals that show improvements to intestinal integrity as well as protecting against the pathology and loss of performance associated with necrotic enteritis in broilers [10, 24, 34, 47]. Future challenge trials will be conducted to determine if the blend of organic acids and botanicals used herein confers protective effects against Clostridium perfringens and necrotic enteritis. Ruminococcaceae families (300 and 500 g/MT) also increased in our study that was accompanied by a decrease in Enterobacteriaceae (in the 500g/MT dose). These data are in agreement with recent studies that also fed diets that incorporated an encapsulated blend of organic acids and essential oils  and phytonutrients . The organic acids and essential oils were different, but the beneficial effects were similar which is also supported by numerous studies using diverse organic acids including, but not limited to, butyric acid , encapsulated benzoic acid , or formic and propionic acids  to enhance the GIT microbiota, poultry health, and performance. Collectively, the data presented herein, along with supporting studies in the literature, demonstrate the importance of targeted release of natural compounds in the poultry GIT to maximize efficacy and potential benefits to the bird. It has been said “increased understanding of how the microbiota interacts with animal hosts will improve microbiome intervention strategies to mitigate production losses” . This statement becomes even more critical as antibiotic use is further curtailed and restricted within the poultry industry, and the present study begins to understand the host-microbiome interaction in the presence of natural antibiotic alternatives.
As with any laboratory experiment, there are limitations that prohibit the inclusion of all variables encountered on the farm. One limitation is the results are qualitative which does not take into consideration cell counts and 16S ribosomal DNA (rDNA) copy number. Different populations can contribute varying copy numbers of 16S rDNA to the analysis; therefore, using quantitative methods will become more important and commonplace as microbiome studies evolve and technologies advance . Additionally, the ability to utilize long read technology will also become necessary to truly understand microbial shifts due to treatment, instead of sequencing small variable regions, such as V3 or V4 that is common today. Despite these limitations, the observed changes in beta diversity will remain consistent and are indicative of potentially optimal microbiota changes. Further, this study demonstrated that shifts in dispersion and mean, as analyzed by ANISOM, occurred by treatment. This type of metric will also stand the test of time and prove essential in delineating the biological role of the microbiota and how it is affected by treatment.
Future studies considering the impact of the biochemical and/or metabolites produced in each compartment of the GIT would provide additional mechanistic insight. Studies in the literature show changes to the microbial populations could diffuse outward or that the metabolites are further transformed by downstream microbial populations impacting colonization by foodborne pathogens such as Salmonella [50, 51]. Dietary supplementation with organic acids and botanicals significantly lowers Salmonella  and Campylobacter  colonization in market-age broilers. Though not considered in those earlier studies, it is possible that changes to the GIT microbial populations while the bird is developing could have contributed to the observed decreases in Salmonella and Campylobacter colonization, but additional studies are required to confirm this hypothesis. Studies support there is compartmental activation of the microbiota; but ultimately it will be the resulting physiological effects within the different compartments as they carry out their specific biological processes [42, 52] that will have the greatest impact. While feed efficiency, nutrient absorption, enzymatic activity and other GIT health indicators were not measured in this study, future studies will determine if these parameters are directly impacted by the microbial population in each compartment of the GIT.