Ureolytic bacteria are key organisms in the rumen producing urease enzymes to catalyze the breakdown of urea to ammonia for the synthesis of microbial protein. However, little is known about the diversity and distribution of rumen ureolytic microorganisms. The urease gene (ureC) has been the target gene of choice for analysis of the urea-degrading microorganisms in various environments. In this study, we investigated the predominant ureC genes of the ureolytic bacteria in the rumen of dairy cows using high-throughput sequencing. Six dairy cows with rumen fistulas were assigned to a two-period cross-over trial. A control group (n = 3) were fed a total mixed ration without urea and the treatment group (n = 3) were fed rations plus 180 g urea per cow per day at three separate times. Rumen bacterial samples from liquid and solid digesta and rumen wall fractions were collected for ureC gene amplification and sequencing using Miseq. The wall-adherent bacteria (WAB) had a distinct ureolytic bacterial profile compared to the solid-adherent bacteria (SAB) and liquid-associated bacteria (LAB) but more than 55% of the ureC sequences did not affiliate with any known taxonomically assigned urease genes. Diversity analysis of the ureC genes showed that the Shannon and Chao1 indices for the rumen WAB was lower than those observed for the SAB and LAB (P < 0.01). The most abundant ureC genes were affiliated with Methylococcaceae, Clostridiaceae, Paenibacillaceae, Helicobacteraceae, and Methylophilaceae families. Compared with the rumen LAB and SAB, relative abundance of the OTUs affiliated with Methylophilus and Marinobacter genera were significantly higher (P < 0.05) in the WAB. Supplementation with urea did not alter the composition of the detected ureolytic bacteria. This study has identified significant populations of ureolytic WAB representing genera that have not been recognized or studied previously in the rumen. The taxonomic classification of rumen ureC genes in the dairy cow indicates that the majority of ureolytic bacteria are yet to be identified. This survey has expanded our knowledge of ureC gene information relating to the rumen ureolytic microbial community, and provides a basis for obtaining regulatory targets of ureolytic bacteria to moderate urea hydrolysis in the rumen.
Urea, a non-protein nitrogen for dairy cows, is rapidly hydrolyzed to ammonia by urease produced by ureolytic bacteria in the rumen, and the ammonia is used as nitrogen for rumen bacterial growth. However, there is limited knowledge with regard to the ureolytic bacteria community in the rumen. To explore the ruminal ureolytic bacterial community, urea, or acetohydroxamic acid (AHA, an inhibitor of urea hydrolysis) were supplemented into the rumen simulation systems. The bacterial 16S rRNA genes were sequenced by Miseq high-throughput sequencing and used to reveal the ureoltyic bacteria by comparing different treatments. The results revealed that urea supplementation significantly increased the ammonia concentration, and AHA addition inhibited urea hydrolysis. Urea supplementation significantly increased the richness of bacterial community and the proportion of ureC genes. The composition of bacterial community following urea or AHA supplementation showed no significant difference compared to the groups without supplementation. The abundance of Bacillus and unclassified Succinivibrionaceae increased significantly following urea supplementation. Pseudomonas, Haemophilus, Neisseria, Streptococcus, and Actinomyces exhibited a positive response to urea supplementation and a negative response to AHA addition. Results retrieved from the NCBI protein database and publications confirmed that the representative bacteria in these genera mentioned above had urease genes or urease activities. Therefore, the rumen ureolytic bacteria were abundant in the genera of Pseudomonas, Haemophilus, Neisseria, Streptococcus, Actinomyces, Bacillus, and unclassified Succinivibrionaceae. Insights into abundant rumen ureolytic bacteria provide the regulation targets to mitigate urea hydrolysis and increase efficiency of urea nitrogen utilization in ruminants.
ObjectiveThe objective of this experiment was to investigate the effects of heat stress on milk protein and blood amino acid profile in dairy cows.MethodsTwelve dairy cows with the similar parity, days in milk and milk yield were randomly divided into two groups with six cows raised in summer and others in autumn, respectively. Constant managerial conditions and diets were maintained during the experiment. Measurements and samples for heat stress and no heat stress were obtained according to the physical alterations of the temperature-humidity index.ResultsResults showed that heat stress significantly reduced the milk protein content (p<0.05). Heat stress tended to decrease milk yield (p = 0.09). Furthermore, heat stress decreased dry matter intake, the concentration of blood glucose and insulin, and glutathione peroxidase activity, while increased levels of non-esterified fatty acid and malondialdehyde (p<0.05). Additionally, the concentrations of blood Thr involved in immune response were increased under heat stress (p<0.05). The concentration of blood Ala, Glu, Asp, and Gly, associated with gluconeogenesis, were also increased under heat stress (p<0.05). However, the concentration of blood Lys that promotes milk protein synthesis was decreased under heat stress (p<0.05).ConclusionIn conclusion, this study revealed that more amino acids were required for maintenance but not for milk protein synthesis under heat stress, and the decreased availability of amino acids for milk protein synthesis may be attributed to competition of immune response and gluconeogenesis.
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