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, 30-35]. 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 [33]. 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 segment compared to control-fed chickens [30]. Specifically, the Rap1 signaling pathway was activated compared to ileal samples [30] which could contribute to intestinal homeostasis and enhanced barrier function [36]. 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.
The microencapsulated blend of organic acids and botanicals used herein is recognized by the European Union Commission and European Food Safety Authority ([EFSA]; AviPlus®P EFSA identification number 4d3) for its ability to enhance growth and feed efficiency in healthy chickens. In the current study, there were no weight differences (P > 0.05) between the supplement- and control-fed chicks, but the chicks on the supplemented diets were slightly heavier than the NTC in a dose-dependent manner. The fact that there were no statistical differences in weight is not surprising since the study was terminated at 15 days which is similar to another feed additive study carried out for 14 days [37]. Broiler studies focused on growth and performance are typically carried out to 35-42 days with incremental measures incorporated into the study design [10, 28, 38]; however our findings do suggest early shifts in the jejunal microbial populations and signaling pathways [30] could be contributing to the enhanced growth and performance that was observed during the EFSA approval process. In fact, in a study conducted on broilers fed with incremental doses of the same microencapsulated blend of organic acids and botanicals demonstrated a linear increase of body weight and improvement of feed efficiency starting at 7 days and reaching the maximum effect at 35 days [29]. The performance results were correlated to an increase in Lactobacillus counts in the feces thus suggesting the change in microbial populations as one of the factors contributing to the overall result at the completion of the grow-out. Nevertheless, microbial populations are just one of the factors determining growth performance as there are also important contributions coming from the host, the environment, and the complex group of interactions among these elements. As one study cannot unveil and explain the complexity of all of these interactions, with the present study we wanted to understand the impact of microencapsulated organic acids and botanicals on the jejunum and ileum populations at the early stages of development of broiler chickens irrespective of growth performance at the end of grow-out. The limitation of this kind of approach resided in the lack of correlation between the changes we observed at day 15 days and the impact these might have had on the final performance. To more definitively address the correlation between microbial populations and performance, time course studies with additional biological replicates and increased numbers of chicks need to be conducted through the entire grow-out. Poultry feed additive studies typically focus on one or more aspect of performance (body weight or feed efficiency); however, the objective of the current project sought to follow-up on the earlier kinome study where signaling differences were observed at the tissue level (ileum versus jejunum) which, as we have shown herein, tissue-specific differences were also observed in the microbial populations.
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 [39, 40]. 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, 41]. 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 [42, 43]; 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 [39, 40]. 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 [44]; 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 [16]. 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 [16]. 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 [10]. 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 [45, 46]. The jejunum is the main sight for nutrient absorption in poultry [42], as well as in mammals, and it has been suggested that the jejunum is the most logical site to observe treatment effects [13] 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, 38, 47]. 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 [9]. Additionally, dietary supplementation with thymol has shown to increase Lactobacillus populations in the ileum [24, 47]; 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 [23]. Another natural compound, a green tea component, also resulted in increased Lactobacillus in the jejunum compared to the ileum when fed to chickens [48]. 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 [49]. 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, 35, 50]. Future challenge trials will be conducted to determine if the blend of organic acids and botanicals used herein confers protective effects against Clostridium perfringens-induced necrotic enteritis and what role the GIT microbial populations play in determining disease outcome. 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 [10] and phytonutrients [49]. 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 [38], encapsulated benzoic acid [27], or formic and propionic acids [28] 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” [51]. 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-controlled experiment, there are limitations that prohibit the inclusion of all variables encountered on the farm under commercial conditions. One of the most obvious discrepancies would be the environment where the newly hatched chick is placed. Under commercial conditions chicks would be placed in a house that has been exposed to thousands of chickens compared to an experimental room that is thoroughly disinfected prior to placement of chicks onto clean litter versus some commercial settings where chicks would be placed on used litter. Clearly, these differences would likely impact the outcome of a microbiome experiment due to the immediate exposure to the myriad of microorganisms found in a poultry house; however, this does not diminish our findings as clear compartmentalization of the microbial populations between the ileum and jejunum were observed. One approach to mitigate this experimental limitation would be to place the chicks on used litter to more realistically mimic the early GIT colonization seen under commercial conditions; however, this approach introduces uncontrollable variables making reproducibility of results difficult. With respect to the microbiome analysis approach that was employed, one of the limitations 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 [52]. 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; however, the approaches we employed are widely accepted and are common practice today [48, 53]. Despite the above-mentioned 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. It should also be noted that these limitations exist with all currently conducted microbiota studies that are not commercially derived, with many of these limitations existing for decades. Yet, studies such as the one conducted here are considered academically sound and important for the industry. While microbiota studies will become more advanced as technology and bioinformatics improves, the importance of academically derived studies independent of field conditions will always be important and relevant.
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 [54, 55]. Dietary supplementation with organic acids and botanicals significantly lowers Salmonella [31] and Campylobacter [32] 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 [46, 56] that will have the greatest impact.
Finally, although outside the scope of the current manuscript, the authors recognize the importance of determining feed efficiency, nutrient absorption, and other GIT functionality traits as predictors and contributors to the final growth performance and on-farm profitability.