Spatial Variation of the Gut Microbiota in Broiler Chickens as Affected by Dietary Available Phosphorus and Assessed by T-RFLP Analysis and 454 Pyrosequencing

Abstract

Molecular fingerprinting and sequencing based techniques have been widely used to characterize microbial communities. Terminal restriction fragment length polymorphism (T-RFLP) and 454-pyrosequencing were used to determine the microorganisms present in the different sections of the chicken gastrointestinal tract (GIT) (crop, jejunum, ileum and caeca). Broilers fed with diets differing in phosphorous (P) and calcium (Ca) as well as in phytase levels were used to study the microbiota of the upper and lower part of the GIT. A database with terminal restriction fragments (T-RF) of the most important organism present in the different gastrointestinal sections was constructed. The analysis revealed a distinct microbial assemblage on each section. Regardless of the diet, crop, jejunum and ileum were mainly colonized by Lactobacillaceae, and caeca were the most diverse site. The correlation between Lactobacillus crispatus and L. reuteri was positive in the crop, but negative in the jejunum. In crop samples, higher P and Ca levels led to a shift in the abundance of L. reuteri and L. crispatus to L. salivarius and L. taiwanensis whereas in the ileum supplementation of phytase favored L. salivarius and L. taiwanensis but resulted in decreased abundance of L. crispatus. Both methods were correlating significantly, being T-RFLP a reliable fingerprinting method to rapidly analyze large numbers of samples in a cost-effective and rapid manner. Results are easy to interpret with no need of deep bioinformatics knowledge and can be integrated with taxonomic information.

Insights into Broilers' Gut Microbiota Fed with Phosphorus, Calcium, and Phytase Supplemented Diets

“…P is, however, a non-renewable resource that is expected to be depleted in the next 100 years (Shastak and Rodehutscord, 2013). Phytate, an organic source of P contained in plant seeds and plant-based diets for broilers, is a principal source of P for the animal, but it has the disadvantage of not being easily accessible by broilers (Witzig et al, 2015; Zeller et al, 2015). The P availability of plant-based diets can be improved by supplementing the diets with phytase, an enzyme that increases P digestibility and reduces P excretion (Witzig et al, 2015).…”

“…Phytate, an organic source of P contained in plant seeds and plant-based diets for broilers, is a principal source of P for the animal, but it has the disadvantage of not being easily accessible by broilers (Witzig et al, 2015; Zeller et al, 2015). The P availability of plant-based diets can be improved by supplementing the diets with phytase, an enzyme that increases P digestibility and reduces P excretion (Witzig et al, 2015). In consequence, the amount of calcium (Ca) and P required in diet formulation can be reduced following release of these two elements from phytate complexes (Zeller et al, 2015).…”

“…They play a crucial role in feed digestion, breakdown of toxins, exclusion of pathogens, stimulation of the immune system, and endocrine activity (Zhu et al, 2002). Several studies have analyzed the microbiota from specific sections of the GIT including the crop, ileum, and caeca (Sekelja et al, 2012; Sergeant et al, 2014; Ptak et al, 2015; Witzig et al, 2015), whereas only a few have focused on the whole GIT (Lu et al, 2003; Sekelja et al, 2012). Nonetheless, it is now known that they are highly connected and should influence up and down-stream the different GIT sections (Stanley et al, 2014).…”

“…Nonetheless, it is now known that they are highly connected and should influence up and down-stream the different GIT sections (Stanley et al, 2014). Most studies have focused on content of the GIT (digesta) samples only (Sekelja et al, 2012; Walugembe et al, 2015; Witzig et al, 2015), ignoring the mucosa communities, that are the closest to the host epithelium (Collado and Sanz, 2007). Epithelium attached microbial communities have biological roles that should be characterized.…”

“…The crop, the section where feed is temporally stored and fermentation activities initiate, is highly dominated by Lactobacillus species (Stanley et al, 2014; Witzig et al, 2015). The ileum, where nutrients are absorbed, is mainly colonized by Lactobacillus species and also by partially characterized bacteria with butyrate producing activities, such as Clostridium, Streptococcus , and Enterococcus (Stanley et al, 2014).…”