Abstract :
[en] Urea has been used in the diets of ruminants as a non-protein nitrogen source. Ureolytic bacteria are key organisms in the rumen producing urease enzymes to catalyze the breakdown of urea to ammonia (NH3), and the NH3 is used as nitrogen for microbial protein synthesis. In the rumen, hydrolysis of urea to NH3 occurs at a greater rate than NH3 can be utilized by rumen bacteria, and excess ammonia absorbed into blood may be harmful to the animals. Nowadays, little is known about the information of ureolytic microorganisms in the rumen, and the changes that occur in the rumen microbial and host metabolites induced by urea nitrogen have not been fully characterized. ‘Omics’ approaches, such as metagenomics and metabolomics have been applied to analyzing rumen microbial community and nutrients metabolism in dairy cows. The objective of this study is to investigate the rumen predominant ureolytic bacteria community and the mechanisms of urea utilization in ruminants using sequencing and metabolomics approaches. Firstly, an in vitro experiment trying to explore the ruminal ureolytic bacterial community was performed. Urea or acetohydroxamic acid were supplemented into the rumen simulation systems as the stimulator and inhibitor for ureolytic bacteria, respectively. The bacterial 16S rRNA genes were analyzed by Miseq sequencing and used to reveal the ureolytic bacteria by comparing different treatments. We found that urea supplementation significantly increased the proportion of ureC genes. The rumen ureolytic bacteria were abundant in the genera of Pseudomonas, Haemophilus, Neisseria, Streptococcus, Actinomyces, Bacillus and unclassified Succinivibrionaceae. Secondly, an in vivo experiment was taken to investigate differences in ureolytic bacterial composition between the rumen digesta and rumen wall based on ureC gene classification. Six dairy cows with rumen fistula were assigned to a two-period cross-over trial. One group was fed a total mixed ration without urea and the treatment group was fed rations plus 180 g urea per cow per day. Rumen bacterial samples from rumen content and rumen wall fractions were collected for ureC gene amplification and sequencing using Miseq. More than 55% of the ureC sequences did not affiliate with any known taxonomically assigned urease genes. The wall-adherent bacteria had a distinct ureolytic bacterial profile compared to the bacteria in the rumen content. The most abundant ureC genes were affiliated with Methylococcaceae, Clostridiaceae, Paenibacillaceae, Helicobacteraceae and Methylophilaceae families. Relative abundance of the operational taxonomic units (OTUs) affiliated with Methylophilus and Marinobacter genera were significantly higher in the bacteria on the rumen wall than that in the rumen content. Thirdly, based on the in vivo experiment, rumen fluid and blood samples were collected and analyzed using nuclear magnetic resonance spectroscopy and multivariate analysis of variance. Concentrations of valine, aspartate, glutamate, and uracil in the rumen, and urea and pyroglutamate in the plasma were increased after urea supplementation. Metabolic pathways include pantothenate and CoA biosynthesis, beta-alanine metabolism, valine, leucine, and isoleucine metabolism in the rumen, and urea and glutathione metabolism in the plasma were significantly increased by urea nitrogen. In conclusion, this study identified significant populations of ureolytic bacterial community that have not been recognized or studied previously in the rumen and provides a basis for obtaining regulatory targets to moderate urea hydrolysis in the rumen. The findings also provided novel information to aid understanding of the metabolic pathways affected by urea nitrogen in dairy cows, and could potentially help to guide efforts directed at improving the efficiency of urea utilization in the rumen.
