Nutraceuticals Induced Structural Changes in Broiler Gastrointestinal Tract Microbiota

Background The effects of nutraceuticals on the modulation of the intestinal microbiota are receiving increased attention however there are scarce number of studies investigating their effects in broiler meat production. The aim of this study was to implement feeding strategies and carry out a comprehensive trial on the interplay between carotenoids, anthocyanins, fructo-oligosaccharides, probiotics and gastrointestinal tract microbiota. Our feeding program was applied on an intensive production system with 1080 broiler Ross 308 flock. Results We observed that nutraceuticals and synbiotics did not affect growth performance remarkably nevertheless, positive correlation was found between body weight and the beneficious Bacteroidales , Corynebacteriales and Pseudomonadales . Nutraceuticals were shown to boost broiler intestinal diversity and differentially enriched Lactobacillus , Enterococcus , Streptococcus and Escherichia-Shigella in core microbiome during the different stages of broiler rearing. Furthermore, diet supplemented with different nutraceuticals was shown to increase the number of unique beneficial bacteria ( Faecalibacterium, Akkermansia ). Conclusion We concluded that nutraceutical-based feeding strategies can offer a promising, green approach for intensive poultry rearing by improving the health and production of livestock. We believe that the conversion of food industrial waste would result more sustainable bioactive component rich forages which invention could be implemented by other commercial antibiotic-free animal production system providing safe and quality meat.

Four beta diversity heatmaps were generated by measuring Bray-Curtis, Jaccard, Weighted-and Unweighted Unifrac distances (Fig. 3f) between the different experimental groups in relation to age and diet. Distance-based dissimilarity matrices showed that flock development exerted remarkable influence on overall community variations thus a gradual increase in community diversity was accompanied with increased heterogeneity of the GIT microbiota.

Significant shifts in community taxonomy were revealed due to age and diet
To further discover key taxa representing significant shifts among study parameters and experimental settings, differentially abundant linear discriminant analysis (LDA) effect size (LEfSe) method was used to perform class comparisons among study groups. We found 22 bacterial clades which were significantly enriched with respect to age and diet (Fig. 5

Diet significantly impacts the composition of the broiler GIT microbiota
We unravelled diet induced and age-related compositional differences at phylum, class, genus and species taxonomic ranks through the broiler production by cataloguing the GIT microbiota of the six experimental groups. Under our experimental settings we identified in total 7 phyla, 12 classes, 20 orders, 31 families and 60 genera. The phyla Firmicutes (89.53%±2.94), Proteobacteria (7.39%±2.90) and Bacteroidetes (1.44%±0.73) were the most predominant accounting for the 32.79%±42.62 of the sequence reads followed by, Actinobacteria, Proteobacteria and Verrucomicrobia (Fig. 6a). Diet related differences in the Firmicutes to Bacteroides ratios (F/B ratio) may reflect alterations in (poly)saccharide utilisation of flocks. The highest log2 F/B ratio was detected in CS2 birds 7.14 (92% vs 0.65%) by the inclusion of β-glucan, while it proved to be the lowest in anthocyanin treated samples 4.89 (83.6% vs 2.8%). Epsilonbacteraeota, Tenericutes and Verrucomicrobia were also detectable, but with very low abundances (≤ 1%). Anthocyanins were shown to increase the proportion of Proteobacteria. Furthermore, anthocyanin treatment was also associated with the highest proportion of Verrucomicrobia (0.48%±0.002). Appreciable age and diet related alterations were found in the proportions of the dominant classes; Clostridia (24.36%±5.9), Bacteroidia (1.49%±0.73), Gammaproteobacteria (6.95%±2.82) (Fig. 6b). As it is shown, diet exerted a strong effect on the distribution of the 6 most abundant classes. Bacilli (62%±6.2) dominated in all experimental groups with the highest relative frequency in animals receiving β-glucan (CS2 71.1%±2.2) whilst the lowest frequencies were measured due to anthocyanin treatment (TS6 53.8%±0.82). Apparently, we observed low variations in the relative abundances of Bacilli among group CS2 (71.1%±2.2), TS3 (58.42%±1.1), TS4 (67%±0.8) and TS5 (61.63%±1.8). Clostridia and Gammaproteobacteria were represented with high proportions (C: 24%±5.9 and G: 7%±2.8). Diet did not impact Bacteroidia (1.4%±0.7). Effective levels of nutraceuticals and probiotics as feed supplements are important resource providers of SCFA production. In the present study the effects of nutraceuticals on SCFA producing genera were also investigated. As shown in Fig. 6c anthocyanins exerted a positive effect on the abundance of Faecalibacterium during finisher feeding period. Significantly lower abundances were observed in 31-day old birds receiving FOSs (TS4 0.88%±0.299) in comparison to birds fed with basal forage (CS1 9.26%±1.8). In general, FOSs, synbiotics and carotenoids were able to exert positive effect on the abundance of the genus Faecalibacterium in chickens older than 31 days.
The drastic alterations in relative frequencies of Lactobacillus (55%±19) were rather age-than diet related. Similarities in abundance patterns were observed between animals fed with basal forage and those receiving β-glucan, carotenoids and synbiotics representing fluctuating trends in relative frequencies. On the contrarily, FOSs and anthocyanins represented an increasing tendency by reaching day 31. Elevated levels of Lactobacillus during the starter (day 1-9) and finisher (day 32-42) phases of production might be associated with the antipathogenic characteristics of the members of this genus. Lactobacillus alvi represented increments in anthocyanins treated samples (0.11%±0.2 vs. other groups 0.015%±0.09). The levels of Lactobacillus salivarus and L. aviarius proved to be relatively high in all experimental groups. L. salivarius which is known to exhibit a protection against Salmonella and other pathogen colonization showed enrichment in the control animals (CS1, CS2; 15%±1.2 vs. treatment groups; 7.5%±2.2), whereas L. aviarius showed remarkable increments due to synbiotics (TS5; 3%±0.2) and anthocyanins (TS6; 7%±0.9). Noticeably, a rise in the genus Campylobacter and the bacterial diarrheal gastroenteristis-causing C. jejuni (TS6 0.4%±0.022 vs others 0.06%±0.02) was shown in anthocyanin fed animals without affecting chicken welfare. In the case of the butyrate producing genera Subdoligranumlum and Butyricoccus similarly to Lactobacillus alternating (ascending and descending) frequencies were observed presenting higher proportions during the first quarter of broiler rearing. Furthermore, anthocyanin treatment (TS6 2.89%±1.05) significantly increased the abundance of Subdologranulum. The genera Streptococcus, Bacteroides, Blautia and Ruminococcus were not prevalent in chicks younger than 7 days. Anthocyanins and synbiotics were able to elevate the levels of Bacteroides notably in both 7-day and 19-day old chicken. Noticeably, in the case of Streptococcus and Blautia the highest proportions were registered on day 31. By the end of the meat production anthocyanin treatment significantly increased the abundance of Blautia (TS6 1.32%±0.4 vs CS1 0.1%±0.18) and Ruminococcus (TS6 0.1%±0.05 vs CS1 0%±0) in comparison to controls.

Comparison Of Diet Induced Structural Modulations
We explored remarkable alterations in family taxonomic data due to carotenoids, FOSs, synbiotics and anthocyanins. A composite heat map was created to pronounce distortions in the relative frequencies normalized to the data of control animals fed with non-supplemented basal forage (Fig. 7). During the pre-starter phase, we observed remarkable increments in Bifidobacteriacea, Ruminococcacea and Erysipelotrichacea due to synbiotics and anthocanins. Also, greater abundances were seen in Ruminococcacea and Erysipelotrichaceae, Clostridiacea and Lachnospiracea in anthocyanin (TS6) challenged animals. Nutraceuticals uniformly decreased the level of Staphylococcacea and Leuconostocacea in comparison to controls. In FOSs challenged 19-day old birds remarkable increments were shown in Barnesiellace, Brevibacteriaceae, Bacteroidaceae and Clostridiacea accompanied by decrements in Bifidobacteriacea, Burkholderiacea. During the grower phase (day 22-31) of meat production appreciable shifts were manifested due to carotenoids (TS3) representing large increments in Bifidobacteriacea, Barnesiellacea and decrements in Aerococcacea, Enterococcacea, Clostridiacea, Peptostreptococcacea and Moraxellacea. Further remarkable declines were evidenced in Dermabacteriacea, Planococcacea, Staphylococcacea, Leuconostocacea, Clostridiacea and Pseudomonadacea due to β-glucan (CS2). In 31-day old animals solid increment in Campylobacteriacea, Planococcaceae and Pseudomonadacea and cutbacks in Bacteroidacea, Helicobacteriacea were registered due to anthocyanins. By the finisher phase of meat production impressive diminutions were encountered in Brevibacteriacea in all of our treatment groups. Enrichments in Helicobacteriacea through FOSs, synbiotics and anthocyanins were detected. Also, during finisher (day 32-42) feeding period a rise was detected in Akkermansia due to β-glucan, synbiotics and anthocyanins.

Nutraceuticals induced structural shifts in comparison to controls
Taxonomic heat trees have been made to generate comprehensive microbial community profiles to represent nutraceuticals induced community shifts in relation to both of the control groups. (Fig. 8). FOSs and synbiotics did not shift the abundance of Proteobacteria and Gammaproteobacteria. On the contrary, carotenoids (TS3) decreased, while anthocyanins (TS6) increased remarkably their proportions. Noticeably, anthocyanins decreased while carotenoids, FOSs and synbiotics aggregated taxa of the class Bacteroidia. We observed a rise in the phylum Tenericutes as per carotenoids (TS3) and synbiotics (TS5). Depletion was found in the class Alphaproteobacteria in relation to β-glucan (CS2) supplemented diet. Carotenoids (TS3) and anthocyanins (TS6) decreased the relative abundance of the family Lactobacillacea. In the case of the treatment groups a slight increment was observed in Enterococcacea frequencies. Appreciable increase was detected in Clostridium due to FOSs (TS4), synbiotics (TS5) and anthocyanins (TS6).

Comparative Metagenomics Provide Insights Into Interplay Of Taxa
To identify diet influenced mutual interconnections within broiler intestinal microbiota comparative metagenomic analysis was performed which involved the comparison of the family and genus frequency data. We estimated the extent to which genera tend to change together. Relative proportions of taxa were correlated in terms of Spearman's method (Fig. 9). We managed to identify families and genera which were notoriously present among all of our experimental groups with highly similar correlations according to the coefficient values and the directions of associations. The strongest negative correlations between possible opportunistic genera such as
Our prior aim was to develop and apply natural feed additives which can stimulate broiler GIT health, without deteriorating the meat production parameters. The feeding program of this trial was applied according to the normatives widely used in Ross 308 chicken production in Hungary [52]. This technology was designed to achieve high weight gain while producing quality meat. We obtained, that under our experimental conditions diet enriched in FOSs, anthocyanins and synbiotics did not alter growth performance during the production stages supporting the estimations of other data [57,91,92]. Furthermore, based on our findings plant derived nutraceuticals have been shown to strengthen the positive correlations between body weight gain and the beneficious Bacteroidales, Campylobacteriales, Corynebacteriales and Pseudomonadales associated with increased absorption of nutrients through the improvement of the integrity of the intestinal epithelia [78,[93][94][95].
Spore forming probiotic Bacillus species associated with increased body weight, were only attainable in broiler feces receiving β-glucan and anthocyanins (data not shown). It is worth to mention, that by the beginning (prestarter) and by the end (finisher) of the feeding periods of broiler meat production anthocyanins increased significantly the levels of the beneficial bacteria such as Lachnospiraceae, Ruminococcaceae associated with improvements in feed conversion [81], (FCRs; day 7 TS6: 0.26 ± 0.04 vs CS1 0.19 ± 0.04, day 40 TS6: 1.87 ± 0.3 vs. CS1 1.26 ± 0.3). We did not capture gains in frequencies of Lactobacillacea and Bifidobacteriacea which were previously reported to enhance the utilization of fructooligosaccharides in FOSs challenged chicken [47,[96][97][98][99][100][101]. Furthermore, our data did not confirm that the implementation of probiotics in poultry diet correlates with enhanced growth during production period which might be explained by a number of different environmental and genetic factors [54,58].
The intricate interconnection of the genera Lactobacillus [102][103][104][105][106] Enterococcus [107,108], Bifidobacterium [109,110], Clostridium [111], Bacteroides [112], Peptostreptococcus [113] regulates primary bile salt synthesis and secondary bile salt metabolism of the host [114]. Growth-promoting mechanism of most subtherapeutic dose antibiotics can be related to decreased activity of the bile salt hydrolase (BSH) enzyme that catalyses deconjugation of bile salts [115]. Certain Lactobacillus species (such as L. salivarius) are the main suppliers of the enzyme BSH [116]. The noticeable decrease in the Gram-positive intestinal Lactobacillales and Clostridium and Gram-negative Bacteroides due to anthocyanins might also be associated with alterations in bile biotransformation due to the decreased level of deconjugated bile salts through which microbiota exerts a negative impact on host fat digestion and utilization. Based on our data however, we did not observe significant remission in the gain rate of the anthocyanins treated broiler which can be explained by considering the intricate metabolic potential of the GIT microbiota (TS6 day 40: 2590 g vs. CS1: 2758 g). Furthermore, in certain concentrations bile salts are associated with antimicrobial effects trough disrupting bacterial membranes, denaturing proteins, causing oxidative damage to DNA, and controlling the expression of certain eukaryotic genes involved in host defence [117]. Corynebacteriaceae is known to correlate with elevated triglyceride level and weight gain [118]. Nutraceuticals significantly increased Corinebacteriaceae in 40-day old chicken, without important changes in growth performances in comparison to control groups.
The intestinal epithelial layer forms the major barrier between host and environment. Especially, species belonging to the phylum Proteobacteria are reported to increase epithelial cell death and mucus degradations [119]. Only some specialized bacteria such as; Clostridiaceae, Lactobacillaceae, Helicobacteraceae and Enterococcaceae are capable to adhere to mucus layer suggesting that these bacteria have a pivotal role in maintaining the gut intestinal barrier integrity [120][121][122]. Mucin degrading Akkermansia which have been previously shown to lower visceral fat deposits are associated with decreased body weight gain rate [123]. Furthermore, the presence of mucin-degrading bacteria is associated with intestinal health, due to competitive exclusion of other bacteria which adhere less effectively to the mucosal surface [121,123]. Based on our data, the increase in the abundance of Akkermansia due to anthocyanins treatment decreased body weight moderately (TS6 2590 ± 280 g vs CS1 2758 ± 264 g) and FCR (TS6 1.36 ± 0.19 vs CS1 1.06 ± 0.3) in comparison to controls.
Bacterial saccharolytic fermentation can transform non-digestible dietary carbohydrates into bioactive molecules associated with positive health outcomes and regulation of the appetite [124]. Among a variety of metabolites produced by the beneficious gastrointestinal tract microbiota short-chain fatty acids (SCFAs) received increased attention because of their important role in disease prevention and recovery [125]. Both Bacteroides and Firmicutes are associated with SCFA synthesis. According to data, increments in Firmicutes can be associated with an increase in nutrient absorption, whereas an elevation in Bacteroidetes correlates usually with enhanced hydrolysis of glycogen, starch and polysaccharides [1,21,43,75,[126][127][128]. Firmicutes to Bacteroidetes ratio (F/B ratio) is important for the optimal nutrition of the host. In this study F/B ratio was the lowest in anthocyanins challenged animals resulting into lower body weight in comparison to controls. There was an increase in Bacteroides in 19-day old flock due to synbiotics that might also correlate with enhanced activity of polysaccharide metabolism since members of this genus are generally associated with degradation of starch and glucan [69]. Acetate and propionate are mainly produced by Bacteroidetes while Firmicutes are the main butyrate supplier [55].
Members of the Bacteroidetes are associated with alpha-amylase, alpha-1,2 mannosidase and endo-1,4-betamannosidase production being involved in the metabolism of starch and other polymeric substances [129]. Synbiotics based diet was shown to favor of the occurrence of the important propionate producer Bacteroides [130,131]. The highest ratios for Bacteroides dorei, B. gallinaceum were detected in samples receiving carotenoids, synbiotics and anthocyanins. By the end of the production period, anthocyanins ameliorated the levels of Bacteroideacea and Barnesiellacea usually linked to more efficient intestinal absorption of components as described previously [83] that might be suggestive of improvements in growth parameters, however, this was also not strengthened by our data. Effects of nutraceuticals manifested in gains in the proportion of the butyrate producer Lachnosiracea and Ruminococcacea in 40-day old chicken. For the colonocytes butyrate is an important energy source which is largely metabolized in the epithelial mucosa [132]. Anthocyanins were shown to favor for the relative enrichment of important butyrate producers; Eubacterium and Faecalibacterium [62,64,124,133] while FOSs and synbiotics proved to be propulsive for the increment of the genus Clostridium associated with beneficiary effects on chicken GIT health [134] in broilers especially during the finisher feeding period.
We also investigated the effects of different dietary supplements on the community complexity through the production of Ross 308 Gallus gallus forma domestica. Therefore, alpha diversity indices (Chao-1, Faith's PD, Shannon and Simpson) were monitored during the four feeding periods (pre-starter, starter, grower, finisher). Based on our results, distinctive differences were observed in GIT microbiome richness among our experimental groups. In general, a tendency representing a gradual increase in richness and evenness was captured by reaching a plateau around day 31. By the end of the broiler productive lifespan a steep decrease was seen in alpha diversity indices in broilers receiving basal diet nevertheless, anthocyanins significantly increased the Chao-1, Shannon and Faith's phylogenetic diversity indexes in comparison to controls. To the best of our knowledge this is the first study investigating the effect of anthocyanins on poultry GIT community diversity. Based on our estimations, FOSs supplemented diet increased alpha diversity indexes (Chao-1 and Faith's phylogenetic diversity indexes) which were consistent with the results reported by Shang et al. [53]. Furthermore, in accordance to a previous study [135], we found that carotenoids did not exert significant effects on community complexity. Probiotics are also increasingly applied to animals especially in poultry industries [61,136]. In agreement to our findings, Baldvin et. al also reported that probiotics based dietary supplementations exerted a positive effect on community diversity [137]. According to our findings, β-glucan supplementation did not have remarkable influence on community diversity. Similarly, to previous reports our data indicated that the composition of the broiler GIT microbiota diversifies remarkably as the GIT microbial population becomes more complex in ageing broiler [21,61,62,138]. Increase in the community alpha diversity made symbiotic communities more discordant which was also supported by Bray-Curtis, Jaccard, Weighted-and Unweighted Unifrac distances. Noteworthy, the present study revealed that appreciable beneficial effects of nutraceuticals manifested mostly by the end of the broiler productive lifespan, as the diversity started to decrease. This may suggest that dietary supplementation has a lesser impact on a more diverse symbiotic microbiota. A higher microbial diversity is commonly related to a healthier host status, whereas the lack of sufficient diversity in a microbial community structure has been associated with different intestinal diseases [22,[139][140][141][142][143][144][145]. Furthermore, imbalance of the gut microbiome composition often leads to the elimination of subset of beneficial bacteria, while the abundance of pathogenic bacteria increases, in conjunction with significant loss of diversity [146].
One of our paramount purposes was the portrayal of GIT core microbiota of livestock. A combined age-related view was achieved at the phylum, order and genus taxonomic ranks to unravel the intricate interconnections of core bacteria at different stages of broiler Ross 308 production. This showed that broiler GIT microbiota was dominated by the two core phyla; Firmicutes (93%±6.9) and Proteobacteria (6.9%±0.9). The order Clostridiales being concordant with a substantial amount of beneficious SCFAs represented conflicting abundances among the control and treatment groups. Interestingly, during the grower period of the broiler production, a remarkable decline (7.3%±0.3) in its presence was observed as per administration of plant derived nutraceuticals. Notorious members of the potential pathogen genera Clostridium, Campylobacter, Staphylococcus, Fusobacterium and Helicobacter have also beneficial physiological effects on various biological responses by synthesizing essential vitamins and micronutrients; thiamine pyrophosphate, riboflavin, nicotinamide, pantothenic acid, biotin, tetrahydrofolate, neurotransmitters; biogenic amines (TMAO), secondary bile acids, lipopolysaccharides for the host [83,147,148]. Furthermore, certain members of the genus Clostridium are known polyphenol producers, possessing antioxidant activity and decreasing inflammation [149]. As such, in the case of Clostridium the lowest ratios were observed in birds treated with carotenoids and anthocyanins. Lipoglycans of Clostridium and Enterococcus spp. are known to trigger inflammatory responses and insulin resistance [150].
One can note, that identifying symbiotic and dysbiotic taxa is not a straightforward task and there are no obvious "good or bad guys" in the complex microbial communities. However, it is essential to consider the problem of contamination of livestock both for sanitational and economic reasons [97]. In our experimental rearing system with 1080 animals the mortality proved to be very low, 0.56% nonetheless no significant differences in lethality patterns were observed between our experimental settings. We aimed to estimate the effect of the nutraceuticals on the susceptibility of the host to pathogen colonization. Therefore, we managed to investigate how nutraceuticals can shift the abundance of Campylobacter, Fusobacterium, Enterococcus, Eggerthella, Helicobacter and Clostridium associated with potential zoonotic strains such as Salmonella enterica, Clostridium difficile, Campylobacter jejuni, and Helicobacter pylori causing enteric diseases and subsequent contamination of poultry products.
Previous studies reported about decreased Campylobacter and Clostridium colonisation measured in broiler fed with fructans [58]. According to our data, the proportion of Campylobacteriaceae was significantly decreased in 40-day old animals receiving immunostimulants in comparison to controls. Furthermore, with the exception of the finisher feeding period, we measured increasing Clostridiacea concentrations to control group (log2 abundance difference 3.12 ± 1.36) in FOSs treated animals. We did not find appreciable differences in the abundance of the genera Fusobacterium (P < 0.99), Eggerthella (P < 0.99) due to different dietary settings, whereas in the case of the Campylobacter spp. a notable increment was registered in birds fed with synbiotics, while carotenoids and anthocyanins were able to decline Helicobacter spp. in comparison to basic controls (TS3 0.69%±0.02, TS6 0.189 ± 0.01 vs. CS1 1.1%±0.05). Campylobacter jejuni was traced in all of our experimental groups. Interestingly, in anthocyanins fed chicken a noticeably increment was registered for the bacterial diarrheal gastroenteritis-causing C. jejuni (TS6 0.009%±0.022 vs others 0.001%±0.06 P < 0.673) without affecting chicken welfare. Of note, C. jejuni can also be involved in the maintenance of intestinal epithelial integrity and the modulation of anti-inflammatory and antitumor effects [53,87,151].
The final two weeks of the growing period of the broiler production systems are associated with elevated mortality and production losses due to localized or systemic bacterial infections. In the poultry industry, besides being commensal and playing important role in the digestion of carbohydrates and proteins some of the members of the genus Clostridium are important pathogens [99] that colonize the gastrointestinal (GI) tract of chicken causing necrotic enteritis [47][48][49]. Infections caused by avian pathogenic Clostridium perfringens and Escherichia coli are among the relevant economically significant problems appreciably deteriorating poultry industry worldwide [98,99]. Although the specific mechanisms have not been fully elucidated, by supporting host immunity, phytonutrients rich in antioxidants can reduce pathogenic stress [152]. The Gram-negative, rodshaped, opportunistic pathogen Alcaligenes faecalis which can trigger infections by colonizing the respiratory tract [153] was not traceable in broiler receiving either β-glucan or nutraceuticals. Being potential pathogens the genus Bacteroides also encode a high number of proteins involved in polysaccharide and monosaccharide metabolism, decrease colonic pH, improve the function of the epithelial cells [60]. In maximizing flock productivity, beside the gastrointestinal tract microflora, water quality, feed, temperature, ventilation and humidity are relevant external factors that influence success of meat production performance.
The most widely used probiotics are members of the relevant acetate producer genus Lactobacillus [45,154] which have also been reported to affect gut health of poultry positively by reducing inflammation, directly modifying intestinal morphology and controlling enteric bacterial infections through regulating mucin composition [29,32,71,106,136,155]. In this trial remarkable enrichments in lactic acid bacteria were identified in phase grower to finisher (day 31 -day 40) due to carotenoids. During the whole experiment, robust relative abundances were observed in FOSs supplemented animals which can be related to the specific enzymatic activities associated with the oligosaccharide transport system of Lactobacilli [80,156]. These data are consistent with the results of other studies reporting Lactobacillus as a major beneficial bacterium that showing increments in broilers fed with fructans [55,59]. In the case of the control samples remarkably elevated levels were measured for Lactobacillus salivarius which can be associated with enhanced induction of antiinflammatory responses in chicken in comparison to treatment groups receiving immune modulators in the form of herbal extracts such as; carotenoids, anthocyanins, and health promoting prebiotics (fructo-oligosaccharides) and synbiotics providing synergistic effect on the gut health [154]. Furthermore, the age-related oscillating patterns in the genus Lactobacillus might be congruent with the deconjugated bile acid concentrations in broiler chicken [107,157]. Both human and animal studies found an association between the accumulation of lactic acids and different disease states such as colitis and gut resection [158,159]. In our study, taxonomy heat trees represented, that anthocyanins remarkably decreased the relative abundance of the family Lactobacillacea.
Apparently, β-glucan, FOSs and anthocyanins based dietary supplementations represented highly similar mutual interconnections among the most relevant genera. The most pronounced negative correlations between butyrate-producer genera such as Butyricoccus, Ruminococcus and lactic acid-producing Staphylococcus, Streptococcus, Enterococcus and Lactobacillus have been revealed in anthocyanins treated animals. This might be associated with improvements in epithelial intestinal barrier functions by decreasing lactic-acid build-up and increasing osmotic load [160]. In synbiotics fed animals the strong negative correlations between the lactate and acetate producing Bifidobacterium, and other lactic acid producing genera such as Lactobacillus (P < 0.003), Streptococcus (P < 0.004) and Staphylococcus (P < 0.003) allude to the intricate interconnections of synbiotics microbiota. Furthermore, evidences showed that some butyrate producers depend on exogenous acetate to butyrate conversion, which implies that a reduction in acetate producer bacteria can be associated with a decrease in intestinal butyrate levels [160]. In the case of animals fed with non-supplemented diet the genus Lactobacillus which was previously identified with poor feed conversion showed strong negative correlations with Bacteroides, Faecalibacterium improving metabolic efficiency and reducing colonization by undesirable microbes [54,80,154,158,161].

Conclusions
This feeding trial was devoted to improve our knowledge about the interplay between carotenoids, fructooligosaccharides, anthocyanins, synbiotics and the broiler gastrointestinal tract microbiota. Based on our scientific data the following main conclusions can be drawn: i) A tendential increase was measured in broiler GIT community diversity as chicken aged, by reaching a plateau around day 31 of the grower period followed by a sharp decline in alpha diversity metrices. Noticeably, these deteriorating parameters were ameliorated by treating birds with FOSs, synbiotics and anthocyanins. ii) Great emphasis was also placed how the taxonomy data correlate with enhanced bird performance. Based on our observations synbiotics expressed the strongest positive correlations between body-weigt gain and the order Campylobacteriales, Corynebacteriales, Micrococcales and Pseudomonadales. iii) The symbiotic broiler Ross 308 microbiota was also deciphered. Considering the 50% core taxonomy data FOSs, synbiotics and anthocyanins were shown to exert the most emphasized community shifts in comparison to controls by registering the most drastic community shifts especially during the pre-starter and starter periods. iv) In general, Firmicutes, Bacteroidetes, Proteobacteria, Gammaproteobacteria, Alphaproteobacteria, Erysipelatoclostridium, Enterobacteriacea, Burkholderiace, Moraxellacea, Streptococcacea and Delftia were identified as key taxa representing significant shifts in community taxonomy due to nutraceuticals. v) We sorely investigated the alterations in relative frequencies of commensal beneficial, short-chain fatty acid producer bacteria and conditioned pathogens. The Firmicutes to Bacteroides ratio proved to be the highest in β-glucan, and the lowest in anthocyanin treated animals. Coincidently, anthocyanins were shown to increase Proteobacteria, Verrucomicrobia, Ruminococcus, Blautia whilst Bacilli which dominated across all experimental groups represented remarkable increments due to βglucans. Generally, FOSs, synbiotics and anthocyanins exerted a positive impact on Faecalibacterium, which difference was more pronounced by the end of broiler rearing. Impressive alterations in Lactobacillus were mostly age related with carotenoids and anthocyanins representing abatements in frequency data. Carotenoids were shown to increase Alistipes, Bifidobacteriacea, Barnesiellacea while reduce Enterococcacea and Clostridiacea. In general, nutraceuticals consistently decreased the level of Staphylococcacea and Leuconostocacea. vi) Finally, comparative metagenomics was performed to identify mutual interconnections among taxa. Streptococcus, Staphylococcus, Escherichia-Shigella and Rombutsia showed strong positive correlations with each other, whereas exerted strong negative correlations with Ruminococcus, Subdoligranulum and Ruminococcacea. Also, thorought all of our experimental groups, strong positive correlations were observed among relevant SCFA producers, such as Blautia, Faecalibacterium, Bacteroides and Christenellacea by noticing that in anthocyanain treated samples, Blautia whilst in β-glucan treated samples Bacteroides showed no correlations with any of these genera. Given its complexity, this trial is particular, which underpins the health benefits of nutraceuticals as potential dietary adjuncts for intensive, antibiotic-free meat-production system.

Birds and Housing
A total of 1080, one-day-old Ross 308 mixed-sex broilers were used from a commercial hatchery in Hungary. The experiment was carried out on the experimental farm of University of Debrecen. All broilers were placed in the same barn. Chickens were kept in floor pens covered with wood shavings in a thermostatically controlled house at a stocking density of 650 cm 2 /bird. Temperature was 32 °C at placement and gradually decreased by 1.5 °C/week. The birds were exposed to light according as follows: 23L:1D during the first 7 days, 20L:4D between 8 − 28 days and 23L:1D between 29 − 42 days (L = light, D = dark).

Experimental Design And Dietary Treatments
The one-day-old Ross 308 hybrid chickens were randomly placed into 6 experimental groups (3 pens/treatment, 60 birds/pen). The experiment was started at 1 day of age and lasted until 42 days of age. Each group was fed one of the following 6 diets: Control Set1 (CS1), basal diet without any added supplements; Control Set2 (CS2), basal diet including 0.5% β-glucan; Treatment Set3 (TS3), basal diet including 0.5% carotenoids; Treatment Set4 (TS4), basal diet including 0.5% FOS; Treatment Set5 (TS5) basal diet including 0.5% synbiotics; Treatment Set 6 (TS6), basal diet including 0.5% anthocyanins. Broilers were fed with a commercial maize-soybean based basal diet (BD) free of antibiotics according to four phase feeding period: pre-starter 1-9 days, starter 10-21 days, grower 22-31 days, and finisher 32-42 days. All diets were fed in mash form. The components and nutritional composition of BD are given in Table 1. The composition of nutrients of each basal diet was planned to satisfy nutritional requirements of chicken according to National research council. Feed and water were available ad libitum during the entire experiment. Broilers were weighed at 1, 10, 21, 32, and 42 days of age. As growth performance parameters, average body weight (BW), average daily gain (ADG) and average daily feed intake (ADFI) were calculated. Mortality was monitored, and it was low (0.56%) so no veterinary interventions were required.

Determination Of Natural Feed Additives
Carotenoids (TS3) supplement was determined as Remenyik et al. [162] and Csernus et al. [52] Carotenoids were extracted from Hungarian red sweet pepper powder (in 1-5 g) using dichloroethane:acetone:methanol as solvent mixture in 2:2:1ratio. The mixture was stirred in an ultrasonic water bath for 30 minutes and purified through a Munktell-292 filter paper (VWR International, Debrecen, Hungary). Fructooligosaccharides supplement (TS4) was determined as Csernus et. al. [52] Hungarian red sweet pepper was also applied to extract fructooligosaccharides (FOSs) with high arabino-galactose content. To assess the composition of oligosaccharides HP 5890 Gas chromatograph was applied with SP-2380 capillary column (30 m x 0.25 mm, 0.2 µm). Samples were lyophilized and extracted with trifluoracetic acid:acetic acid:water in 5:75:20 proportion as solvent. Oligosaccharides were turned into alditol-acetate. After reduction step, sugars were shifted to sugar alcohols (alditols), which remove interfering isomers and anomers. Reduction was performed with NaBH4 at alkine pH. Acetylation was also performed with acetic anhydride in pyridine. The feed gas was nitrogen at 1.2 mL/min flow rate. The injector temperature was set to 300 ºC and split ratio was 1:20. Flame Ionization Detector (FID) was used for identification of oligosaccharides. The GC profile and the identified monomer units of oligosaccharides are involved in additional file [see Additional file 2] The synbiotics supplement (TS5) contained probiotics (Bifidobakterium bifidum, B.infantis, B.lactis

Sample Collection
Stool samples were collected on day 7, 19, 31, 40 of age. In every treatment group 6 broilers (3 pullets and 3 cockerels) were marked and faecal samples were collected from them during the whole experimental period. Pooled faecal samples were also collected in the case of all our experimental groups. Stool samples were collected freshly into specific, sterile, DNase free stool transportation bowls and were immediately placed on ice for maximum 3 hours. Not processed samples were kept at -80 °C until further use.

Sample Preparation And Mechanical Cell Lyses
Bacterial cell suspensions (BS) were prepared from 7-7 g broiler stool samples. 7-7 ml of sterile PBS buffers (Thermo Fisher Scientific, Maryland, USA) were added to the samples and homogenized for 4 min (by vortexing at 350 RPM) [164]. The samples were centrifuged for 5 min at 500 x g. Supernatants were collected and the washing step was repeated 2 times. Supernatants were centrifuged for 20 min at 13.000 x g. Finally, the supernatants were discarded, and the bacterial pellets were dissolved in 3 ml of sterile PBS buffer. 1 ml aliquots of BS were added to PowerBead Tubes (Qiagen, Hilden, Germeny) for mechanical cell lyses. Bacterial cell disruption was performed with MagNa Lyser Instrument (Roche Applied Sxciences; Penzberg, Germeny) set to 5000 x rpm for 30 seconds.

Dna Extraction
Total bacterial genomic DNA was extracted with conventional isolation method. 800 µl sample lysate was mixed with 800 µl of phenol:chloroform:isoamyl alcohol (25

Sequence Processing And Analysis
The Illumina BaseSpace software was used to demultiplex the paired end reads and construct FASTQ files. The sequencing data were analysed using the Quantitative Insight Into Microbial Ecology (Qiime 2, ver 2019.7) [165]. The presence of adapter sequences (CTGTCTCTTATACACATCT) were checked and trimmed from the 3' end of the reads with Cutadapt Software integrated in the Qiime 2 pipeline. DADA2 software was used for quality trimming, filtering and for chimera removal. Sequences were clustered into ASVs, with 97% similarities in sequences [166].
The trimming parameters were set as follows: for the forward reads 1 bases were cropped from the start and the length was set to 300 bases; for the reverse reads 9 bases were cropped from the start of the reads and the length was set to 223 bases.

Bioinformatic Analyses
Multiple sequence alignment was performed with the Mafft software [167], and reads were taxonomically classified using Naïve Bayesian classifier trained on the Silva (ver132) [168] reference database by selecting mapping points according to the forward-reverse primer set that was used for amplifying the 16S rRNA gene's V3-V4 regions of the bacterial community (341F, 806R). Phylogenetic trees were constructed with FastTree plugin [169]. The QIIME2 pipeline was applied to perform alpha and beta diversity tests. For sample normalization a 11 500 read depth was set. In the case of alpha diversity Shannon's index [170], Faith's phylogenetic diversity index [171], Simpson evenness [172], and Chao-1 index [173] were calculated in the QIIME2 pipeline. For beta diversity analysis weighted/unweighted UNIFRAC distances [174] and Bray-Curtis dissimilarities [175] were measured. Alpha diversity differences were compared using the Kruskal-Wallis test. Beta diversity group significances were calculated with Permutational multivariate analysis of variance (PERMANOVA) pseudo F statistical test. QIIME2 artifact files were exported from the pipeline and converted to TSV files which were used with different visualization packages. Heatmaps were generated in Python (ver3.6.5) with Seaborn package; area, donout plots were constructed with pandas and matplotlib packages. Boxplots, violin plots and line plots were constructed using GraphPad Prism statistical software. R (ver 3.6.2) was used to visualize bubble plots and polar plots. Differential heat tree was created with the metacoder R package [176]. In the case of differential heat trees differences were determined using a Wilcoxon rank-sum test. LEfSe analysis was performed with bioBakery tools developed by Huttenhower lab [177]. Spearman correlation matrices were calculated and visualised with R statistical software using the corrplot package (https://github.com/taiyun/corrplot).

Statistical Analysis Of Growth Performance
The main effects of the bioactive compounds on growth performance was analyzed using one-way analysis of variance (one-way ANOVA), Tukey's multiple comparison test was conducted at significance level of P < 0.05.

Availability of data and materials
All sequence data used in the analyses were deposited in the Sequence read Archive (SRA) (http://www.ncbi.nlm.nih.gov/sra) under SUB7398678 Sample IDs, meta data and corresponding accession numbers are summarized in Supplementary figure S1.

Ethics approval
Sampling procedures were carried out in accordance with the ethics committee's approved guidelines (DEMAB/12 − 7/2015).

Consent for publication
Not applicable.

Competing interests
The authors declared that they have no competing interests.

Funding
This study was financially supported by the GINOP-2.3.2-15-2016-00042 project of the Széchenyi 2020 Programme given by the European Union and the Hungarian Government.

Figure 1
Sampling and treatment strategies and effects of natural compounds on growth performance of broiler chickens. Baseline statistics represent the pivotal indicators of feed efficiency; average daily feed intake (ADFI), and      Differential abundance taxonomic heat trees revealed the effects of nutraceuticals. Metacoder differential heat tree illustrates the variation in microbiome phylotypes between experimental groups. Nodes in the heat tree correspond to phylotypes, as indicated by node labels, while edges link phylotypes in accordance to taxonomic hierarchy. Node size corresponds to the number of OTUs observed within a given phylotype. To visualize the effects of dietary supplementation on microbial composition community heat trees were made to represent the effects of the following dietary treatment; CS1 negative control (BD with no dietary supplement), dietary treatments were provided as mash feed; CS2 positive control (BD incl. 0.5% of β-glucan), TS3 (BD incl. 0.5% of carotenoids), TS4 (BD incl. 0.5% of FOSs); TS5 (BD incl. 0.5% of synbiotics), TS6 (BD incl. 0.5% of anthocyanins). The annotated tree on the left functions as a map for the unlabelled trees. Coloured taxons represents the extents of log2 differences in taxa abundances.

Supplementary Files
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