The objective of this project is to evaluate the ruminal microbial diversity involved in the ruminal degradation of soybean meal, cottonseed meal, DGGS and glutenose, and correlate it with ruminal degradation of DM, CP and protein fractions of each ingredient. In each experimental period, after 20 days of adaptation of the animals to the diet, 112 nylon bags in situ containing 5 g of each protein source will be placed in the rumen of cannulated steers fed with the respective protein source. Four nylon bags will be placed in the animals' rumen in reverse chronological order at 48, 24,16, 6, 4, 2 e 0.5h before being removed and eight non-incubated nylon bags filled with the different ingredients will be used as control (0 h). The relative degradation of the biomass during rumen incubation will be determined by MS analysis according to the AOAC methods and CP by multiplication by 6.25 of the nitrogen determined in the LECO FP-528 nitrogen analyzer. CP insoluble in neutral detergent, CP insoluble acid detergent, non-protein nitrogen and soluble protein content of protein sources will be estimated according to Licitra et al. (1996). The nylon bags recovered from the rumen for microbiological analyzes will be washed with PBS buffer solution and immediately frozen and storage in liquid nitrogen and transported to the laboratory for the extraction of metagenomic DNA. Sequencing of the V4/V5 region of the bacterial 16S rRNA gene will be the method used for the identification and quantification of adhered ruminal bacteria. The results will be analyzed considering a 4 x 4 Latin square design, with measures repeated over time.
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