Activity- and Enrichment-Based Metaproteomics Insights into Active Urease from the Rumen Microbiota of Cattle

Regulation of microbial urease activity plays a crucial role in improving the utilization efficiency of urea and reducing nitrogen emissions to the environment for ruminant animals. Dealing with the diversity of microbial urease and identifying highly active urease as the target is the key for future regulation. However, the identification of active urease in the rumen is currently limited due to large numbers of uncultured microorganisms. In the present study, we describe an activity- and enrichment-based metaproteomic analysis as an approach for the discovery of highly active urease from the rumen microbiota of cattle. We conducted an optimization method of protein extraction and purification to obtain higher urease activity protein. Cryomilling was the best choice among the six applied protein extraction methods (ultrasonication, bead beating, cryomilling, high-pressure press, freeze-thawing, and protein extraction kit) for obtaining protein with high urease activity. The extracted protein by cryomilling was further enriched through gel filtration chromatography to obtain the fraction with the highest urease activity. Then, by using SDS-PAGE, the gel band including urease was excised and analyzed using LC-MS/MS, searching against a metagenome-derived protein database. Finally, we identified six microbial active ureases from 2225 rumen proteins, and the identified ureases were homologous to those of Fibrobacter and Treponema. Moreover, by comparing the 3D protein structures of the identified ureases and known ureases, we found that the residues in the β-turn of flap regions were nonconserved, which might be crucial in influencing the flexibility of flap regions and urease activity. In conclusion, the active urease from rumen microbes was identified by the approach of activity- and enrichment-based metaproteomics, which provides the target for designing a novel efficient urease inhibitor to regulate rumen microbial urease activity.

The Temporal Dynamics of Rumen Microbiota in Early Weaned Lambs

Weaning affects the development of ruminal bacteria in lambs during early life. However, the temporal dynamics of rumen microbiota in early weaned lambs is unknown compared to conventionally weaned lambs. In this study, one group was reared with their dams (control, CON) and conventionally weaned at 49 days (d), while the other lambs were weaned at 21 d (early weaning, EW) using starter. Rumen microbial samples collected at 26, 35, and 63 d were used for next-generation sequencing. Here, we found that the abundance and diversity of rumen microbiota in EW were significantly lower at 26 and 35 d than the CON. Linear discriminant analysis Effect Size (LEfSe) analysis was performed to identify the signature microbiota for EW at these three ages. At 26 d, Prevotella 7, Syntrophococcus, Sharpea, Dialister, Pseudoscardovia, and Megasphaera in the rumen of the EW group had greater relative abundances. At 35 d, the Lachnospiraceae_NK3A20_group was enriched in CON. On 63 d, Erysipelotrichaceae_UCG-002 was abundant in EW. Syntrophococcus and Megaspheaera in EW lambs were abundant at 26 and 35 d, but kept similar to CON at 63 d. The relative abundance of Erysipelotrichaceae_UCG-002 at all-time points was consistently higher in the EW group. In conclusion, early weaning led to a significant decrease in rumen microbiota richness and diversity in the short term. The changes in rumen microbiota are associated with the persistence of weaning stress. The temporal dynamics of relative abundances of Syntrophococcus, Megasphaera, and Ruminococcaceae_UCG-014 reflect the weaning stress over a short period and rumen recovery after early weaning.

Rumen Microbiota Alterations During Ketosis is Associated with the Development of Mastitis in Dairy Cows

Backgroud: Mastitis is the most serious disease endangering animal husbandry, especially dairy farming. Clinical investigations indicated that cows suffering from ketosis have a higher probability of mastitis. Rumen microbiota is closely related to ruminant health. However, it is not clear what role it plays in this process.Results: The microbiota in rumen fluid and milk from ketosis cows were determined by 16S rRNA gene sequencing. The results showed that the richness of bacterial community both in rumen and milk were changed in ketosis cows. The abundance of genus Prevotella, Ruminococcus, Succinivibrionaceae_UCG-001 and Streptococcus in rumen fluid from ketosis cows decreased significantly and were negatively correlated with blood BHBA and milk SCC. In contrast, the abundance of genus Luteimonas, Thermomonas, Christensenellaceae_R-7_group, Rikenellaceae_RC9_gut_group, NK4A214_group, Paracoccus, Acetitomaculum, Prevotellaceae_UCG-003, Deinococcus, Saccharofermentans and Butyrivibrio in rumen fluid from ketosis cows increased significantly and were positively correlated with blood BHBA and milk SCC. In addition, the abundance of F082 and Thermomonas were increased, while the abundance of genus Acinetobacter and UCG-005 were reduced both in milk and rumen fluid in ketosis cows than healthy cows. Conclusions: Ketosis in dairy cows is capable of inducing mastitis. The rumen microbiota of ketotic cows changed significantly and is associated with the development of mastitis. Targeting rumen microbiota regulation may be a promising strategy to prevent metabolism disorder and its secondary diseases in dairy cows.

The Effects of Non-Fiber Carbohydrate Content and Forage Type on Rumen Microbiome of Dairy Cows

The main objective of our current study was evaluating the effects of NFC supplementation and forage type on rumen microbiota and metabolism, by comparing microbial structures and composition among samples collected from cows fed AH (alfalfa-based diet), H-NFC (CS-based diet with high NFC) and L-NFC (CS-based diet with low NFC) diets. Our results show that microbial communities were structurally different but functionally similar among groups. When compared with L-HFC, NFC increased the population of Treponema, Ruminobacter, Selenomonas and Succinimonas that were negatively correlated with ruminal NH3-N, and urea nitrogen in blood, milk and urine, as well as significantly increasing the number of genes involved in amino acid biosynthesis. However, when compared to the AH group, H-NFC showed a higher abundance of bacteria relating to starch degradation and lactate production, but a lower abundance of bacteria utilizing pectin and other soluble fibers. This may lead to a slower proliferation of lignocellulose bacteria, such as Ruminococcus, Marvinbryantia and Syntrophococcus. Lower fibrolytic capacity in the rumen may reduce rumen rotation rate and may limit dry matter intake and milk yield in cows fed H-NFC. The enzyme activity assays further confirmed that cellulase and xylanase activity in AH were significantly higher than H-NFC. In addition, the lower cobalt content in Gramineae plants compared to legumes, might have led to the significantly down-regulated microbial genes involved in vitamin B12 biosynthesis in H-NFC compared to AH. A lower dietary supply with vitamin B12 may restrict the synthesis of milk lactose, one of the key factors influencing milk yield. In conclusion, supplementation of a CS-based diet with additional NFC was beneficial for nitrogen conversion by increasing the activity of amino acid biosynthesis in rumen microbiota in dairy cattle. However, lower levels of fibrolytic capacity may limit dry matter intake of cows fed H-NFC and may prevent increased milk yield.

Lovastatin as a supplement to mitigate rumen methanogenesis: an overview

Methane from enteric fermentation is the gas with the greatest environmental impact emitted by ruminants. Lovastatin (Lv) addition to feedstocks could be a strategy to mitigate rumen methane emissions via decreasing the population of methanogenic archaea (MA). Thus, this paper provides the first overview of the effects of Lv supplementation, focusing on the inhibition of methane production, rumen microbiota, and ruminal fermentation. Results indicated that Lv treatment had a strong anti-methanogenic effect on pure strains of MA. However, there are uncertainties from in vitro rumen fermentation trials with complex substrates and rumen inoculum.Solid-state fermentation (SSF) has emerged as a cost-effective option to produce Lv. In this way, SSF of agricultural residues as an Lv-carrier supplement in sheep and goats demonstrated a consistent decrease in ruminal methane emissions. The experimental evidence for in vitro conditions showed that Lv did not affect the volatile fatty acids (VFA). However, in vivo experiments demonstrated that the production of VFA was decreased. Lv did not negatively affect the digestibility of dry matter during in vitro and in vivo methods, and there is even evidence that it can induce an increase in digestibility. Regarding the rumen microbiota, populations of MA were reduced, and no differences were detected in alpha and beta diversity associated with Lv treatment. However, some changes in the relative abundance of the microbiota were induced. Further studies are recommended on: (i) Lv biodegradation products and stability, as well as its adsorption onto the solid matter in the rumen, to gain more insight on the “available” or effective Lv concentration; and (ii) to determine whether the effect of Lv on ruminal fermentation also depends on the feed composition and different ruminants.

Sex Differences in Rumen Fermentation and Microbiota of Tibetan Goat

Background: Gut microbiota play an important role in maintaining host metabolism, immune system and health, while sex, genotype, diet and health have certain effects on composition of gut microbiota. Therefore, in order to explore the sex differences in the structure and function of rumen microbiota in Tibetan goat, the study analyzed the sex differences in rumen fermentation parameters, rumen microbiota and the expression of genes related to VFAs transport in Tibetan goat. Results: The results showed that the content of acetic acid in the rumen of rams was significantly higher than that of ewes (P <0.05), and propionic acid and butyric acid were higher than that of ewes, but the difference was not significant (P >0.05); The expression of VFAs transport related genes DRA, AE2, MCT-1, NHE1, NHE2 in rumen epithelium of ewes was significantly higher than that of rams. Analysis of the composition and structure of rumen microbiota showed that there were significant sex differences in structure of rumen microbiota, and the abundance of rumen microbiota in ewes was higher than that in rams (P>0.05). At the phylum level, Firmicutes and Bacteroidetes were the dominant phyla of Tibetan goat, while Firmicutes was significantly higher in ewes than in rams (P<0.05). At the genus level, the relative abundance of Fibrobacter, Ruminococcus_1 and Pyramidobacter in ewes was significantly higher than that in rams (P<0.05). The function prediction results showed that Replication, recombination and repair, RNA processing and modification were mainly enriched in ewes (P<0.05). Conclusions: Correlation analysis revealed significant associations of some rumen microbiota with the fermentation product VFAs and VFAs transport-related genes. It is concluded that rams and ewes have a strong ability for fermenting and metabolizing when adapting to the plateau environment, which provides a certain sex reference basis for Tibetan goat adaptation to the plateau environment.

Combined Supplementation of Probiotics and Enzymes Improves Performance and Regulates Rumen Microbiota in Fattening Goats

Background: The aim of this study was to explore the effects of growth performance, slaughter performance, serum biochemical, immune and antioxidant indexes and rumen microflora fed with a basal diet (CON group), added B. subtilis and B. licheniformis (PRO group), supplemented with B. subtilis, B. licheniformis and enzyme preparations (COM group) on fattening goats.Methods: 39 male goats were randomly divided into 4 groups with 13 individuals in each group for feed period of 80 d. Goats were fed as follows: CON diet, PRO diet with B. subtilis and B. licheniformis, and COM diet with B. subtilis, B. licheniformis and compound enzymes.Results: ADFI of COM group significantly increased compared with CON group and PRO group (P < 0.01), as well as COM group and PRO group dramatically promoted ADG versus with CON group (P < 0.05). As a consequence, the body weight of fattening goats in the COM group was predominantly higher than that in the CON group (P < 0.01). In addition, the PRO group and COM group enhanced the TNF-α (P < 0.05) and IL-10 content (P < 0.01) in the serum. No differences were observed in serum biochemical and antioxidant indexes of three groups (P > 0.05). Likewise, the GR values of PRO group and COM group were noteworthy improved in comparison with CON group (P < 0.01). The VFA contents in rumen fluid were insignificantly different (P > 0.05). COM group also enriched the relative abundance of Proteobacteria compared with CON group and PRO group (P < 0.05). Nevertheless, the relative abundance of Actinobacteria decreased of PRO group and COM group in rumen fluid microorganisms (P < 0.05). Apparently, COM group significantly enriched nitrogen metabolism, glycolysis and TCA cycle (P < 0.05), whereas nucleotides biosynthesis was notably reduced (P < 0.05).Conclusion: The combined feed of probiotics and enzymes had more profound effects than probiotics feed. Consequently, supplementation with B. subtilis and B. licheniformis and enzymes in the basal diet of fattening goats, which could improve growth performance, slaughter performance, immunity and accommodate rumen microbiota.