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.

Feeding with high-concentrate diets increases the risk of subacute ruminal acidosis (SARA). This experiment was conducted to evaluate whether supplementing a phytogenic feed additive based on L-menthol, thymol, eugenol, mint oil (Mentha arvensis) and cloves powder (Syzygium aromaticum) (PHY) can amend the ruminal fermentation profile, modulate the risk of SARA and reduce inflammation in cattle. The experiment was designed as a crossover design with nine non-lactating Holstein cows, and was conducted in two experimental runs. In each run, cows were fed a 100% forage diet one week (wk 0), and were then transitioned stepwise over one week (0 to 65% concentrate, wk adapt.) to a high concentrate diet that was fed for 4 weeks. Animals were fed diets either with PHY or without (CON). The PHY group had an increased ruminal pH compared to CON, reduced time to pH < 5.8 in wk 3, which tended to decrease further in wk 4, reduced the ruminal concentration of D-lactate, and tended to decrease total lactate (wk 3). In wk 2, PHY increased acetate, butyrate, isobutyrate, isovalerate, and the acetate to propionate ratio compared to CON. Phytogenic supplementation reduced inflammation compared to CON in wk 3. Overall, PHY had beneficial effects on ruminal fermentation, reduced inflammation, and modulated the risk of SARA starting from wk 3 of supplementation.

Salivary secretions are essential for the regulation of digestive processes, as well as rumen and cow health. This research evaluated the effects of the duration of high-grain feeding, and of the time relative to a meal, on salivation, saliva properties, feed bolus characteristics, chewing activity, ruminal and reticular volatile fatty acids, as well as salivary and ruminal pH. Nine nonlactating cannulated Holstein cows were sampled at 1 and 23 d after transition to a 65% grain diet (short term and long term, respectively). Both before and after a controlled meal (2.5 kg of dry matter, offered over 4 h), unstimulated saliva was taken orally for composition analysis. Stimulated salivation and feed boli characteristics were evaluated by collection of ingesta from cardia during 30 min. Chewing and ruminal pH were measured during the controlled meal and for a total of 6 h thereafter. Results from unstimulated saliva showed no effect of the duration of high-grain feeding on bicarbonate, phosphate, total proteins, mucins, lysozyme, and buffer capacity, but increased osmolality at the long term. Lysozyme activity did not differ with high-grain feeding duration, but tended to be lower after the meal. In contrast to short-term-fed cows, the long-term-fed cows increased both meal consumption and feed bolus size, but decreased chewing and feed ensalivation (5.2 vs. 4.6 ± 0.50 g of saliva/g of dry matter), and had lower pH of the stimulated saliva (7.00 vs. 6.67 ± 0.076). These cows also had decreased chewing index (66.5 vs. 45.4 min/kg of neutral detergent fiber), and despite the increase in stimulated saliva buffer capacity (0.027 vs. 0.039 ± 0.006), mean ruminal pH decreased (6.31 vs. 6.11 ± 0.065) during ad libitum feeding. Both in the rumen and reticulum, the concentration of total volatile fatty acids was lower and propionate proportion was higher at the long term. Linear regression analyses revealed a positive influence of the flow rates of salivary bicarbonate and phosphate on ruminal pH during the short term. For every 1-mol increment in the flow of bicarbonate or phosphate, ruminal pH increased by 0.062 or 0.439 units, respectively. Overall, salivary buffers are key determinants of ruminal pH regulation, especially during short-term grain feeding. However, in the long term, ruminal pH drop during ad libitum feeding was stronger, and this effect seems to be exacerbated by increased feed bolus size, accompanied by reductions in feed ensalivation, stimulated saliva pH, and chewing index.

Untargeted metabolomics requires robust and reliable strategies for data processing to extract relevant information form the underlying raw data. Multiple platforms for data processing are available, but the choice of software tool can have an impact on the analysis. This study provides a comprehensive evaluation of four workflows based on commonly used metabolomics software tools: XCMS, Compound Discoverer, MS-DIAL, and MZmine. These tools were applied to a dataset derived from bovine saliva samples spiked with small polar molecules analyzed by anion exchange chromatography coupled to high resolution mass spectrometry. The analysis revealed significant differences in the number and overlap of detected features, with only approximately 8 % of the features included in all four peak tables. Among the overlapping features, MS-DIAL demonstrated the greatest similarity to manual integration, while XCMS and MZmine also performed well. In contrast, Compound Discoverer had issues to reliably integrate high baseline peaks. This study also explores various post-processing strategies, including missing value imputation, transformation, scaling, and filtering. The assessment of missing values indicated that they primarily originated from low abundance, making imputation with small values the most effective approach. No clear evidence suggested that transformation is necessary for downstream statistical analyses. Auto scaling emerged as the most suitable strategy for data scaling. Low thresholds for blank filtering were found to be the most effective in enhancing data quality. The optimization of filtering thresholds required a careful balance to remove unnecessary information while retaining vital data. This work provides an overview of commonly applied strategies in untargeted metabolomics analysis, emphasizing the importance of careful workflow selection and optimization. It serves as a resource for refining data processing strategies to achieve accurate and reliable results, while also offering fresh insights into the challenges encountered throughout the untargeted metabolomics processing pipeline.

In cattle, proper rumen functioning and digestion are intimately linked to chewing behavior. Yet, high grain feeding impairs chewing activity, increasing the risk of subacute ruminal acidosis and dysfermentation. This study aimed to screen 9 different phytogenic compounds for their potential to modulate chewing activity, meal size, rumino-reticular short-chain fatty acids (SCFA), and pH during consumption in a first daily meal and shortly thereafter in cattle fed a grain-rich diet. Treatments were control (total mixed ration without phytogenic) or addition of a phytogenic compound at a low or high dose. Phytogenic compounds and doses (all in mg/kg) were angelica root (6.6 and 66), capsaicin (10 and 100), gentian root (6.6 and 66), garlic oil (0.3 and 3), ginger extract (40 and 400), L-menthol (6.7 and 67), mint oil (15.3 and 153), thyme oil (9.4 and 94), and thymol (5 and 50), for the low and high groups, respectively. Before the start of the screening experiment, cows were fed to reach subacute ruminal acidosis conditions, confirmed with the time of ruminal pH <5.8 being 655 ± 148.2 min/d. During the screening experiment, the treatments were offered in a controlled meal (2.5 kg of DM for 4 h) as part of the daily diet with 65% concentrate. Each treatment was tested in 4 of the 9 cannulated Holstein cows using an incomplete Latin square design. Ruminal and reticular fluids were sampled before and after each treatment, and data collected before the meal were used as covariates. Chewing and ruminal pH were monitored during the treatment, followed by 2 h of complete feed restriction, and then 4 h of ad libitum feed intake without phytogenic. Data showed that supplementation of angelica root tended to linearly increase rumination time immediately after the first meal when feed was restricted (27.3, 41.9, and 42.6 ± 5.99 min for control, low and high groups, respectively). Capsaicin increased eating time (43.6, 49.4, and 66.4 ± 4.93 min) during consumption but did not affect ruminal total SCFA or mean ruminal pH. Garlic oil reduced the concentration of reticular total SCFA (75.7, 71.3, and 60.1 mM) and tended to decrease ruminal acetate-to-propionate ratio (2.50, 1.78, and 1.87 ± 0.177) with no effect on ruminal pH. The L-menthol affected reticular total SCFA quadratically (76.1, 64.9, and 81.0 ± 4.22%), and ruminal pH responded quadratically when feed was reintroduced ad libitum (6.0, 6.3, and 6.1 ± 0.07). Mint oil did not affect chewing or total SCFA during consumption, but the low dose increased ruminal pH (6.5, 6.7, and 6.5 ± 0.08). Thyme oil tended to lower the severity of ruminal acidosis. Overall, phytogenic compounds demonstrated distinct dose-dependent effects to beneficially influence chewing behavior, modulate fermentation, and mitigate ruminal acidosis in dairy cows under a high-grain challenge diet.

The aims of this research were to evaluate how prolonged feeding of a high-concentrate diet affects the ruminal degradation kinetics of fiber and starch, and to evaluate the effects of the high-concentrate diet on apparent total-tract nutrient digestibility in dairy cows. We also investigated the dysbiotic effects and the remodeling of the hindgut microbiome with prolonged high-concentrate feeding. Nine Holstein cows were used in 2 experimental periods; in each period, cows were first fed a 100% forage diet (Forage) for 1 week, followed by stepwise adaptation during one week to a high-concentrate diet (HC; 65% concentrate), which was then fed for 4 consecutive weeks. The kinetics of in situ ruminal degradability of grass silage (DM and NDF), corn grain and wheat grain (DM and starch) as well as the apparent total-tract nutrient digestibility were evaluated in the Forage feeding and in wk 4 on HC. Whereas the hindgut microbiome and fermentation profile were evaluated on a weekly basis. Regarding the in situ ruminal degradability due to grain type, the rate of degradation of the potentially degradable fraction of the grain and the effective rumen degradability of wheat grain were greater compared with corn grain. The in situ ruminal degradability of NDF decreased with the HC diet. However, the apparent total-tract digestibility of crude protein, fat, starch, NDF, ADF and NFC increased with HC compared with Forage feeding. In addition, the HC diet increased the concentration of short-chain fatty acids in the hindgut, lowering fecal pH by 0.6 units, which correlated positively with microbial α diversity. This resulted in lower α diversity with HC; however, α diversity (number of ASVs) showed recovery in wk 3 and 4 on HC; in addition, microbial β diversity did not change from wk 2 on HC onwards. Two microbial enterotypes were identified: one for the Forage diet with abundance of Akkermansia and Anaerosporobacter, and another enterotype for the HC diet with enrichment in Bifidobacterium and Butyrivibrio. Overall, results show that major microbial shifts and hindgut dysbiosis occurred in wk 1 on HC. However, the hindgut microbial diversity of cows adapted after 3 weeks of consuming the starch-rich ration. Thus, feeding HC diet impaired fiber degradation in the rumen, but increased apparent total-tract nutrient digestibility. Likely, the forage diet contained less digestible NDF than the HC diet due to greater inclusion of forages with lower NDF digestibility and lower inclusion of more digestible non-forage NDF. Results also suggest that the adaptation of the hindgut microbial diversity of cows observed 3 weeks after the diet transition likely contributed to enhance total-tract nutrient digestibility.