Characterization of microbial intolerances and ruminal dysbiosis towards different dietary carbohydrate sources using an in vitro model.

AIM: This study aimed to characterize the critical points for determining the development of dysbiosis associated with feed intolerances and ruminal acidosis. METHODS AND RESULTS: A metabologenomics approach was used to characterize dynamic microbial and metabolomics shifts using the rumen simulation technique (RUSITEC) by feeding native cornstarch (ST), chemically modified cornstarch (CMS), or sucrose (SU). SU and CMS elicited the most drastic changes as rapidly as 4 h after feeding. This was accompanied by a swift accumulation of d-lactate, and the decline of benzoic and malonic acid. A consistent increase in Bifidobacterium and Lactobacillus as well as a decrease in fibrolytic bacteria was observed for both CMS and ST after 24 h, indicating intolerances within the fibre degrading populations. However, an increase in Lactobacillus was already evident in SU after 8 h. An inverse relationship between Fibrobacter and Bifidobacterium was observed in ST. In fact, Fibrobacter was positively correlated with several short-chain fatty acids, while Lactobacillus was positively correlated with lactic acid, hexoses, hexose-phosphates, pentose phosphate pathway (PENTOSE-P-PWY), and heterolactic fermentation (P122-PWY). CONCLUSIONS: The feeding of sucrose and modified starches, followed by native cornstarch, had a strong disruptive effect in the ruminal microbial community. Feed intolerances were shown to develop at different rates based on the availability of glucose for ruminal microorganisms. SIGNIFICANCE AND IMPACT OF THE STUDY: These results can be used to establish patterns of early dysbiosis (biomarkers) and develop strategies for preventing undesirable shifts in the ruminal microbial ecosystem.

Bovine rumen epithelial miRNA-mRNA dynamics reveals post-transcriptional regulation of gene expression upon transition to high-grain feeding and phytogenic supplementation.

The rumen epithelium has a pivotal role in nutrient uptake and host health. This study aimed to explore the role of microRNAs (miRNAs) in the epithelial transcriptome during diet transition from forage to high-grain feeding and the modulation through supplementation with a phytogenic feed additive. Rumen biopsies were collected from 9 ruminally-cannulated non-lactating Holstein cows fed a baseline forage diet (FD) and then transitioned to high-grain feeding (HG; 65% concentrate on a dry matter basis). Cows were randomly allocated into a control group (CON, n = 5) and a group supplemented with a phytogenic feed additive (PHY, n = 4). MiRNA and mRNA sequencing was performed in parallel and transcripts were analyzed for differential expression, pathway enrichment analysis, and miRNA-mRNA interaction networks. We identified 527 miRNAs shared by all samples of the rumen epithelium, from which, bta-miR-21-5p, bta-miR-143 and bta-miR-24-3p were the most expressed. Six miRNAs were differentially expressed between CON and PHY and 8 miRNAs between FD and HG feeding, which were mainly associated with fat metabolism. Transcriptome analysis identified 9481 differentially expressed genes (DEGs) between FD and HG, whereas PHY supplementation resulted in 5 DEGs. DEGs were mainly involved in epithelium development and morphogenesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with tricarboxylic acid and short chain fatty acid (SCFA) metabolism were enriched in DEGs between diets. MiRNA target prediction and anti-correlation analysis was used to construct networks and identify DEGs targeted by DE miRNAs responsive to diet or PHY. This study allowed the identification of potential miRNA regulation mechanisms of gene expression during transition from FD to HG feeding and phytogenic supplementation, evidencing a direct role of miRNAs in host responses to nutrition.

Unveiling the Bovine Epimural Microbiota Composition and Putative Function.

Numerous studies have used the 16S rRNA gene target in an attempt to characterize the structure and composition of the epimural microbiota in cattle. However, comparisons between studies are challenging, as the results show large variations associated with experimental protocols and bioinformatics methodologies. Here, we present a meta-analysis of the rumen epimural microbiota from 11 publicly available amplicon studies to assess key technical and biological sources of variation between experiments. Using the QIIME2 pipeline, 332 rumen epithelial microbiota samples were analyzed to investigate community structure, composition, and functional potential. Despite having a significant impact on microbial abundance, country of origin, farm, hypervariable region, primer set, animal variability, and biopsy location did not obscure the identification of a core microbiota. The bacterial genera Campylobacter, Christensenellaceae R-7 group, Defluviitaleaceae UCG-011, Lachnospiraceae UCG-010, Ruminococcaceae NK4A214 group, Ruminococcaceae UCG-010, Ruminococcaceae UCG-014, Succiniclasticum, Desulfobulbus, and Comamonas spp. were found in nearly all epithelium samples (>90%). Predictive analysis (PICRUSt) was used to assess the potential functions of the epithelial microbiota. Regularized canonical correlation analysis identified several pathways associated with the biosynthesis of precursor metabolites in Campylobacter, Comamonas, Desulfobulbus, and Ruminococcaceae NK4A214, highlighting key metabolic functions of these microbes within the epithelium.

Physicochemical stressors and mixed alkaloid supplementation modulate ruminal microbiota and fermentation in vitro.

The drop of ruminal pH and heat are common physicochemical stressors challenging ruminal microbiota, nutrient digestion and cattle performance. We characterized the ruminal microbiota and digestive activity in response to different pH (6.0 and 6.6) and temperature (39.5 and 42 °C), as well as established the effective dose of alkaloid supplementation (0, 0.088 and 0.175% of feedstock DM) to modulate ruminal fermentation under these conditions. The acidotic condition decreased microbial diversity and abundances of minor bacterial families whereas most of the highly abundant families like Lactobacillaceae, Prevotellaceae, and Bifidobacteriaceae thrived under the stress. Abundances of all three methanogenic archaea taxa detected increased with heat, as did methane production. However, while Methanomassiliicoccaceae benefited from the low pH, Methanomicrobiaceae diminished and methane production decreased. The low dose of alkaloid addition shifted the fermentation to more propionate and less acetate and the high dose decreased methane and ammonia concentration under the low pH. In conclusion, physicochemical stressors shape the microbial community and function. Mixed alkaloid supplementation facilitates the activity of rumen microbial community under acidotic stress.

Feed Additives Differentially Impact the Epimural Microbiota and Host Epithelial Gene Expression of the Bovine Rumen Fed Diets Rich in Concentrates.

The success of nutritional strategies for the prevention of subacute ruminal acidosis (SARA) and the related microbial dysbiosis still remains unpredictable due to the complexity of the rumen ecosystem. The rumen epimural community, due to proximity, has the greatest opportunity to influence host gene expression. The aim of this study was to determine the effect of two separate feed additives on the rumen epimural community and host epithelial gene expression. Eight rumen cannulated Holstein cows were randomly assigned to one of three feeding groups: autolyzed yeast (AY), phytogenics (PHY) and control (CON) using a 3 × 3 Latin square design. Cows were fed an intermittent SARA model that started with 100% forage diet (Baseline) followed by two 65% concentrate-diet induced SARA challenges (SARAI, SARAII), separated by 1 week of forage only feeding. Rumen papillae samples were collected via the cannula during the Baseline, SARAI and SARAII periods. Microbial DNA was extracted and sequenced targeting the 16S rRNA gene and host RNA was analyzed using RT-qPCR. Analysis of the taxonomic composition at the genera level showed a tendency to increase in the relative abundances of Pseudobutyrivibrio (P = 0.06), Selenomonas (P = 0.07) and significantly increase in SHD-231 (P = 0.01) in PHY treated animals, whereas Succiniclasticum tended to decrease in both PHY and AY treated animals compared to the control. Linear discriminant analysis effect size testing was performed and based on treatment × feeding phase interaction, a number of biomarker genera were identified including the previously identified Succiniclasticum. Supplementation with AY correlated positively with CD14 and DRA expression and negatively to CLDN1, MyD88, and MCT4 expression. Supplementation with PHY showed a negative correlation to CLDN4 gene expression. Anaerovibrio showed the highest positive Pearson correlations to biogenic amines tested in the rumen fluid including putrescine (r = 0.67), cadaverine (r = 0.84), and tyramine (r = 0.83). These results show that supplementing feed additives to high grain diets can have a positive influence on the stability of the epimural populations, and that changes in the epimural community are correlated with changes in host epithelial gene expression.

Betaine addition as a potent ruminal fermentation modulator under hyperthermal and hyperosmotic conditions in vitro.

BACKGROUND: Climatic and dietary shifts predispose ruminal microbes to hyperthermal and hyperosmotic stress, leading to poor fermentation and subsequently adverse effects on ruminant productivity. Betaine may function as substrate, osmolyte, antioxidant, and methyl donor for microbes. However, its effect depends on the extent of microbial catabolism. This study revealed the ruminal disappearance kinetics of betaine and its dose effect on ruminal fermentation during thermal and osmotic stress using a rumen simulation technique. RESULTS: Three different betaine doses were used: 0, 50, and 286 mg L-1 ; each was assigned to two incubation temperatures (39.5 and 42 °C) and two osmotic conditions (295 and 420 mOsmol kg-1 ). Betaine disappeared rapidly within the first 6 h of incubation; however, the rate was lower during hyperosmotic stress (P < 0.05), the stress condition that also suppressed the overall fermentation and degradation of organic nutrients and decreased the bacterial diversity (P < 0.001). During hyperosmotic stress, betaine shifted the fermentation pathway to more propionate (P < 0.05). Betaine counteracted the negative effect of hyperthermal stress on total short-chain fatty acid concentration (P < 0.05) without affecting the composition. Both stress conditions shifted the bacterial composition, but the effect of betaine was minimal. CONCLUSION: Despite its rapid ruminal disappearance, betaine modulated microbial fermentation in different ways depending on stress conditions, indicating the plasticity of the betaine effect in response to various kinds of physicochemical stress. Although betaine did not affect the abundance of ruminal microbiota, the enhanced fermentation suggests an improved microbial metabolic activity under stress conditions. © 2020 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Graded replacement of corn grain with molassed sugar beet pulp modulates the fecal microbial community and hindgut fermentation profile in lactating dairy cows.

High starch lactation diets not only enhance the risk of subacute ruminal acidosis but also of hindgut acidosis, which increases the risk of dysbiosis and the depression of fiber degradation. We recently showed that replacing corn with molassed sugar beet pulp (Bp) improved fiber degradation in high-producing dairy cattle, possibly because of an improvement of rumen and hindgut conditions for microbes by Bp feeding. However, little is known about the effects of high inclusion rates of Bp on hindgut microbes and fermentation. Thus fecal grab samples were taken from 18 high-yielding Simmental cows after 28 d of feeding 3 different levels of Bp (n = 6) for bacterial 16S rRNA amplicon sequencing. In addition, the reticular pH was continuously monitored with indwelling sensors and eating and ruminating behavior was evaluated with noseband sensors. The Bp inclusion rates were 0 g/kg (i.e., no Bp inclusion as control, CON), 120 g/kg (12Bp), or 240 g/kg (24Bp) replacing corn grain and limestone on a dry matter basis. The amount of time spent eating and ruminating was unaffected by Bp level, and the daily fluctuation in the reticular pH was reduced by 25% with Bp inclusion from 0.8 in the CON diet to 0.6 in 24Bp fed animals. Also, the fecal pH tended to increase with dietary Bp inclusion. Fecal acetate production showed a quadratic tendency with the lowest concentration (58.9%) of the total short-chain fatty acid in the 12Bp treatment. Inclusion of Bp up to 24% of the diet decreased the fecal butyrate proportion by 27%. The Shannon diversity index was increased from 5.50 to 8.09 with dietary Bp inclusion indicating increased species diversity. Of the 200 most abundant operational taxonomic units, 25 were increased by dietary Bp inclusion, whereas 15 were decreased and 7 were quadratically affected. The second most abundant group was proposed taxon "CF231" of the family Paraprevotellaceae. Although it accounted for only 2.52% of the operational taxonomic units in the CON diet, it was increased by 64% with dietary Bp inclusion. The largest relative change in the abundance was found for the genus Fibrobacter that increased more than 14-fold from 0.04% (CON) to 0.66% (24Bp). In conclusion, feeding molassed sugar beet pulp as partial substitution of corn up to 240 g/kg is a viable alternative that promotes ruminal and hindgut fermentation by supporting physiological pH and bacterial diversity.

Effects of the supplementation of plant-based formulations on microbial fermentation and predicted metabolic function in vitro.

This study aimed at testing the effects of three different formulations of feed supplements based on three different combinations of plant derived alkaloids, prebiotics, tannins, vitamins and minerals on rumen fermentation and the microbiome in vitro. A Rusitec experiment was conducted in 2 identical runs using a complete randomized design with 3 replicates per treatment resulting in total of 6 treatment combinations (n = 6). Each run lasted 12 d with sampling occurring in the last 5 d. Diets were a standard dairy ration (60:40; concentrate:forage) supplemented with one of 3 different plant-based combinations (PI, PII, and PIII) at a level of 100 mg/l and a non-supplemented control (basal diet, control). Microbial DNA samples were taken on the last day of each run and the 16S rRNA target gene sequenced using Illumina MiSeq technology. The supplementations had no effect on the pH, methane and carbon dioxide production. However, both total SCFA (P = 0.08) and molar concentrations of acetate (P = 0.06) tended to be increased in the treatment groups in comparison to control, with PII having the highest overall values (102.7 mmol/L and 43.3 mmol/L, respectively). Alpha diversity indices Shannon, Simpson and Chao1 showed no effect of supplementations or combinations. The addition of PII increased the relative abundance of Bacteroidetes compared to all other treatments (P = 0.05). Supplementation with plant-based combinations reduced the relative abundance of Pyramidobacter from the family Dethiosulfovibrionaceae in comparison with the control diet (P = 0.05). Evaluation of predicted gene function through PICRUSt analysis showed variation in predicted cellular function and metabolism between bacterial communities supplemented with plant-based combinations compared to the control diet. This shows that the addition of plant-based combinations can have the potential to modulate the metabolic function of rumen microbes, and likely the production of small-sized rumen metabolites, without disrupting the rumen microbial community structure and diversity.