In recent decades, demand for free-range poultry meat has risen in Western societies, as a consequence of increasing consumer awareness of issues such as animal welfare, environmental impact, and food safety issues (1). Free-range rearing systems allow animals to express more natural behaviors and provide access to vegetation and other food sources. In broiler chickens, this is known to improve welfare (2) and potentially improve meat quality, composition, and organoleptic properties (3). Furthermore, free-range rearing can also influence production parameters such as body weight, feed intake, and feed conversion ratio (4). The gut microbiota composition of broilers is thought to be an important determinant of animal welfare, performance, and environmental impact (5). A healthy gut microbiome is essential not only to achieve optimal host performance but also to minimize the risk of zoonotic pathogens contaminating human food and adversely impacting consumer health (6). In general, greater microbial diversity is important to ensure host health status, because this plays a vital role in feed metabolism, immune system regulation, as well as the competitive exclusion of pathogenic organisms (7). Furthermore, several forage products are rich in functional molecules, some of which appear to be promising alternatives to antibiotics given that they contribute to the regulation of intestinal microbiota, enhance growth performance, and improve the immune response (8).

Many factors influence poultry gut microbiota diversity and composition, such as diet, rearing conditions, host genetics, and age (9–11). Age and breeding systems have been identified as key factors contributing to microbial diversity, but further studies are necessary to understand the importance of different rearing systems, particularly free-range systems (1). It will be important to establish a baseline for free-range systems to enable comparisons with factors such as diet and environment. Broiler chicken gut microbiota have been investigated for several years (12), but most efforts have focused on intensive systems of broiler production, and studies on other rearing systems are scarce. Therefore, the aim of this study was to characterize the composition, diversity, and predicted functions of the cecal microbiota community present in free-range broilers over their lifespan using next-generation sequencing (NGS) technologies. The intention was to broaden our understanding of the factors responsible for changing microbiota composition.

The present study examined a free-range system where broiler crossbreeds were reared in a certified brand breeding system in Southern Italy. This alternative system production is characterized by the use of GMO-free feed and the complete absence of antibiotics. It is also characterized by using renewable energy sources, a key point in the sustainability project of this rearing system. Finally, we report how symbiotic and commensal partnerships between bacteria and free-range broiler chicken could contribute to animal health and nutrition ranging from the metabolism of complex carbohydrates to the supply of important micronutrients such as queuosine and its precursors and vitamins.

This study provided a comprehensive sequence-based characterization of the cecal microbiota community of free-range grown chickens before and after their outdoor access period. Although microbiome analysis techniques have been applied to conventional poultry production systems (28, 29), there has been less focus on free-range systems. Reports on the cecal microbial community of broilers older than 42 days are scarce, and it is well recognized that many factors influence poultry gut microbiota composition and intestinal function such as diet, age, host genetics, breeding system, geographical location, and environmental conditions (1). Age-related processes occurring in the broiler lifespan such as feed changes, immune system development, and exposure to extraneous microorganisms result in gut microbiota shifts with advancing age. Studies have established that by day 21 of life, the microflora reaches a point of maturity; however, other production factors such as the beginning of the finisher diet from day 28 of life introduce further selective changes in the broiler gut microbiota, and by day 42 of life, over 200 genera colonize the intestinal tract (30). Therefore, it can be hypothesized that diet and rearing system are the principal variables that influenced the major changes in the cecal microbiota of the two groups. Noteworthy, the microbial community composition was considerably distinct before and after outdoor access, when different types of environmental resources, such as grasses, were introduced into the diet. This study agrees with other studies examining chicken cecal microbiota changes related to age and breeding system (31, 32). It finds that Firmicutes and Bacteroidota represented the dominant phyla of the cecal community regardless of whether they had outdoor access. 1 month after outdoor pasture, two new phyla emerged, namely, Deferribacterota and Synergistota. The changes in gut microbiota at the species taxonomic level, before and after outdoor access, were characterized by the onset of Mucispirillum schaedleri, Bacteroides barnesiae, Bacteroides caecigallinarum, and uncultured bacteria of genera Bacteroides and Synergistes after outdoor access of the chickens. Even if less is known about the role of these bacteria, evidence highlights their potential benefit for the host: bacteria from phylum Synergistota are mostly anaerobic microorganisms found in the animal digestive tracts and also in the soil (33); at present, the role of B. caecigallinarum has not been well and deep reported (34), but B. caecigallinarum represents an acetate and succinate producer (35), and it was demonstrated that it increased after the administration of fermented Momordica charantia juice, mitigating hyperglycemia, hyperinsulinemia, hyperlipidemia, and oxidative stress in diabetic rats (36).

Interestingly, the results obtained showed a clear difference in the assortment of Bacteroides genera between the two groups. The increase of Bacteroides genus in Outdoor microbiota is a potential positive finding due to its beneficial role in growth performance and the inhibition of Clostridium perfringens sporulation by its fermentation products (37), thus preventing the dramatic economic losses related to C. perfringens-associated necrotic enteritis (38).

Based on network analysis, B. barnesiae and an uncultured organism of the genus Synergistes might be proposed as keystone taxa whose presence drives the shift of composition and potential functioning of Outdoor microbiota. The network analysis also showed results of considerable interest regarding H. pullorum. Indeed, as revealed by the mutual exclusion pattern, H. pullorum was present only if B. barnesiae, an uncultured organism of the genus Synergistes, and B. gallinaceum were absent, suggesting these as key species driving the reduction of H. pullorum. In poultry, variable prevalence rates of this emerging food-borne pathogen have been reported from various regions with a range from 4 to 100% (27, 39); nevertheless, free-range and organic breedings have been poorly investigated so far. In our study, the significantly lower abundance of H. pullorum following the period of outdoor access (relative abundance <1% compared with >2% before) suggests that the potential for zoonotic infection (by means of broiler carcass contamination) is less likely. However, it's difficult to establish how the various factors involved in the changes of gut microbiota could have influenced our results, and further research is encouraged to deepen the unclarified aspects. We could hypothesize that the higher diversity in microbial community and the expansion of other taxa associated with a healthy gut could reduce the likelihood of colonization by this pathogen, and potential competitive exclusion strategies to limit the prevalence of H. pullorum might be investigated.

Among the minor phyla associated with a healthy gut, we have highlighted the presence of Deferribacterota and, at the species level, of Mucispirillum schaedleri following the outdoor access period. This observation is in line with other studies describing the microbial community of free-range chickens (6, 40). This bacterium is known to have a mucus-associated niche in the gut, and thus, it can be considered a marker for the health of the surface mucus layer in the distal intestinal tract (41). Mucispirillum schaedleri has also been reported to antagonize Salmonella typhimurium virulence and to protect mice against colitis by interfering with S. typhimurium invasion gene expression and competing for anaerobic electron acceptors (42).

Another very interesting finding of this study is that metagenomic pathway signatures before and after an outdoor access period differed in terms of their functional roles. Specifically, there were differences in a subset of functional roles (signature genes) that are required for de novo vitamin/cofactor biosynthesis. B vitamins are necessary cofactors for several aspects of human and animal metabolisms, including those concerning fat, carbohydrate, and DNA synthesis (43). Based on our findings, the biosynthesis of vitamins, such as B vitamins, also appears to be higher, underlining that the outdoor evolved microbiota of broilers contribute to the vitamin pool of the gut and that the host could benefit to some extent from the vitamin biosynthesis in the microbiota. Vitamins have remarkable antimicrobial activity in vitro as well as in vivo. Among the water-soluble vitamins, B1, B2, and vitamin B12, augmented after outdoor access in this study, were found to be effective, particularly against Gram-positive bacterial pathogens such as Staphylococcus aureus, Staphylococcus epidermidis, and MRSA, showing synergism with several antibiotics (44).

The gut microbiota provide a variety of micronutrients important for human and animal health. Among them, queuosine (Q), a vital micronutrient, synthesized exclusively by bacteria, represents a known and novel product arising from a microbe–host interaction. In bacteria, Q is synthesized de novo. On the contrary, eukaryotes lack the enzymes required for de novo synthesis of Q and hence rely on nutrient sources and additionally on their gut microbiome (45).

Gut microbiota represent a complex ecosystem that develops in close interaction with the host. In terms of Q metabolism, this environment is particularly complex, as specific microbes can be sources (46). Q plays a regulatory role in translation, cell proliferation, stress responses, and cell signaling. Q deficiency correlates with several phenomena and diseases including stress tolerance, cell proliferation, tumor growth, encephalomyelitis, and leukemia. Of importance, our metabarcoding study shows that the microbiota from broilers with a period of outdoor access are likely to have higher biosynthesis levels of Q and its Q precursor, pre Q0. This precursor is a key metabolite recruited in different pathways and used as a precursor of secondary metabolites, such as toyocamycin and sangivamycin, collectively referred to as deazapurines, in Actinomycetes. The nucleoside analog sangivamycin and its closely related nucleoside antibiotic toyocamycin showed a broad activity against arenaviruses, filoviruses, and orthopoxviruses; and an antitumor activity against leukemia and pancreatic cancer (47–49). This important finding suggests that gut microbiota evolved from the outdoor rearing with access to natural dietary sources increase the biosynthesis of Q and related metabolites. It will now be necessary to (i) undertake next-generation sequencing to fully understand the pathway changes, (ii) measure the differences in the concentrations of various Q pathway metabolites using techniques such as liquid chromatography mass spectrometry, and (iii) measure the extent of tRNA Qylation, in order to understand the importance of different rearing conditions.

This study provided new insights into the dynamics of microbial communities and predictions of metabolic functions in broiler chickens from a free-range system. Obviously, as known, these results coming from predictive imputed functions have evaluation limitations and should be interpreted cautiously. Therefore, further investigation, more replicates, microbiome, and metabolome studies are needed to better corroborate this perspective in a clearer view.

By targeting functional genes of chicken gut microbiota, new approaches might be explored to improve the growth performance and productivity of broiler chickens, and strategies promoting nutrient metabolism can be designed by introducing helpful bacteria possessing nutrient digesting genes involved in the maintenance of host health.

Diet and other environmental factors also represent powerful modulators of the gut microbiome, and further investigations are necessary to better understand how we can responsibly harness this inner ecosystem to make animals healthier, reduce antimicrobial use and dependence in livestock, and prevent antimicrobial resistance for an environmental health.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

The animal study was reviewed and approved by Institutional Animal Care and Use Committee of the University of Naples Federico II. Written informed consent was obtained from the owners for the participation of their animals in this study.

LB, LV, and LC contributed to the conception and design of the study, conducted the literature searches, wrote the manuscript, and performed the statistical analysis. LD, BG, and AP critically revised the manuscript. LB, FL, LM, and AF read, revised, and concurred with the final version of the review. All authors have made an intellectual contribution to the work and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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