Protease and phytase supplementation effects on amino acid digestibility and small intestinal microbiota composition in broiler chickens

Great losses of feed proteins in broiler chickens are due to the incomplete digestion on the gastrointestinal tract (GIT). Exogenous protease is included in broilers diet to improve protein digestibility, nitrogen retention and reduce protein waste [1]. In this digestion process the intestinal microbiota plays an important role because converts the ingested substrates into short chain fatty acids, amines, amino acids and other compounds that are beneficial to the host [2]. Thus, the aims of this study were to determine how enzymes supplementation affects precaecal aminoacid digestibility in a dose-dependent manner and if the ileal microbiota composition is altered. Methods: Unsexed Ross 308 hatchlings were placed in 72 pens in groups of 15. A commercial starter diet was provided until experimental diets were used starting on day 14. The nutrient-adequate experimental diets were mainly based on maize and soybean meal. Treatments involved the control diet without enzyme supplementation, protease A (Meiji) at 25 and 200 mg/kg, protease B (Cibenza) at 500 and 4000 mg/kg, protease C (Ronozyme ProAct) at 200 and 1600 mg/kg and phytase (Natuphos E) at 1500 FTU/kg. Diets were in mash form hatch to 21 days of age and offered for ad libitum intake. Birds were asphyxiated on day 21 and the content from the terminal ileum obtained and pooled on a pen basis and stored at -80°C. Determination of amino acid digestibility followed standard procedures. Precaecal aminoacid digestibility was determined and performance data were also analyzed. Ileal total nucleic acids were obtained with a commercial kit and 16S rRNA gene Illumina amplicon sequencing was used to characterize microbial assemblages. Results: When compared to the control, a significant increase in growth and feed efficiency was caused by phytase and the high dosage of protease C. Average precaecal digestibility of essential amino acids was 82 % in the control diet, 84 % with the high dosage of protease C, and 85 % with phytase. Differences from the control diet were significant for most of the amino acids in these two treatments. Other treatments did not cause a consistent effect on amino acid digestibility A total of 1021 OTUs were identified in the whole dataset. Firmicutes was the most abundant phylum across all diets (> 98%). A significant difference of the bacterial profiles at genera level, was observed among the eight treatments (p=0.024). Streptococcus was contributing to this dissimilarity showing higher abundance in low and high dose supplementation of protease B (24% and 30%, respectively), when compared to 13% abundance in protease C also at high and low doses. Lactobacillus was showing the highest abundance across all diets and revealed to be negatively correlated with other genera (p < 0.05). In Protease C, Lactobacillus accounted for 77% and 64% of the total community at low and high dosages respectively, while it was detected with 38% abundance in diet 8, 43% in diet 4 and 56% in protease B at high doses. Uncultured Clostridiaceae 1 was more abundant with protease B at low (20%) and high dose (10%) and phytase (15%) when compared to protease C, where was only detected in lower abundance (1-4%). The genus Clostridium XI was detected in higher abundance in phytase supplementation (13%) followed by 6% abundance in protease C at high level and between 0.4% and 3% in the other diets.Conclusions: It is concluded that the suitability of different proteases to increase the aminoacid digestibility in conventional broiler diets is variable and dose dependent. The different levels of enzyme have an effect on the main bacterial groups probably caused by the substrate supplementation, degradation of certain nutrients or proteolytic activity.