Adaptive responses in short-chain fatty acid absorption, gene expression, and bacterial community of the bovine rumen epithelium recovered from a continuous or transient high-grain feeding.

The feeding of high-grain diets to dairy cows commonly results in lowered pH and ruminal dysbiosis, characterized by changes in absorption dynamics of short-chain fatty acids (SCFA) across the reticuloruminal wall, epithelial function, and the epithelial bacteria community structure. Therefore, the present study evaluated the effect of high-grain feeding persistence on the absorption kinetics of reticuloruminal SCFA, gene expression in the rumen epithelium, and the associated shifts in the epithelial bacteria in cows recovering from either a long-term continuous high-grain feeding model or a long-term transient high-grain feeding model. In a crossover study design, 8 nonlactating Holstein cows were fed 60% concentrate either continuously for 4 wk (continuous) or with a 1-wk break in the second week of the high-grain feeding (transient). After the high-grain feeding, all animals were fed a diet of 100% forage (recovery) for an additional 8 wk. Rumen papilla biopsies and SCFA absorption measurements were taken at the start of the trial (baseline), after the 4-wk high-grain feeding (49 d), after 2-wk recovery forage feeding (63 d), and after 8-wk recovery forage (105 d). Absorption of SCFA was determined in vivo using the washed and isolated reticulorumen technique. Rumen papillae biopsies were used for adherent bacterial DNA and host RNA extraction. The epithelial bacteria were determined using Illumina MiSeq (Microsynth AG, Balgach, Switzerland) sequencing of the 16S rRNA gene. No significant effects of the high-grain feeding model were seen for bacterial diversity. However, bacterial diversity increased with time spent in the recovery forage feeding period regardless of feeding model. The relative abundance of Acidobacteria phyla and Acetivibrio spp. increased when animals were fed a transient high-grain feeding model. A trend toward increased CLDN4 expression was observed in the continuous model. Furthermore, there were interactions between feeding model and sampling day for gene targets CD14, DRA, NHE2, NHE3, and MCT2. When comparing length of recovery, in the continuous model increased relative absorption of SCFA was sustained at 63 d but dropped to baseline measurements at 105 d. A similar pattern was found with the transient model but it did not reach significance. The only gene target that was found to significantly correlate to relative absorption of SCFA was DRA (correlation coefficient ≤ -0.41). Whereas, genera Alkalibaculum, Anaerorhabdus, Coprococcus, and Dethiobacter all showed positive correlations to gene targets for pH regulation (NHE2 and NHE3) and SCFA uptake (MCT1) but negative correlations to SCFA absorption. We conclude that while the rumen absorption and epithelial bacteria were able to recover to baseline levels after 8 wk of forage feeding, the time needed for re-establishment of homeostasis in host gene expression is longer, especially when high-grain feeding is interrupted.

Epimural bacterial community structure in the rumen of Holstein cows with different responses to a long-term subacute ruminal acidosis diet challenge.

Subacute ruminal acidosis (SARA) is a prevalent metabolic disorder in cattle, characterized by intermittent drops in ruminal pH. This study investigated the effect of a gradual adaptation and continuously induced long-term SARA challenge diet on the epimural bacterial community structure in the rumen of cows. Eight rumen-cannulated nonlactating Holstein cows were transitioned over 1 wk from a forage-based baseline feeding diet (grass silage-hay mix) to a SARA challenge diet, which they were fed for 4 wk. The SARA challenge diet consisted of 60% concentrates (dry matter basis) and 40% grass silage-hay mix. Rumen papillae biopsies were taken at the baseline, on the last day of the 1-wk adaptation, and on the last day of the 4-wk SARA challenge period; ruminal pH was measured using wireless sensors. We isolated DNA from papillae samples for 16S rRNA gene amplicon sequencing using Illumina MiSeq. Sequencing results of most abundant key phylotypes were confirmed by quantitative PCR. Although they were fed similar amounts of concentrate, cows responded differently in terms of ruminal pH during the SARA feeding challenge. Cows were therefore classified as responders (n = 4) and nonresponders (n = 4): only responders met the SARA criterion of a ruminal pH drop below 5.8 for longer than 330 min/d. Data showed that Proteobacteria, Firmicutes, and Bacteroidetes were the most abundant phyla, and at genus level, Campylobacter and Kingella showed highest relative abundance, at 15.5 and 7.8%, respectively. Diversity analyses revealed a significant increase of diversity after the 1-wk adaptation but a decrease of diversity and species richness after the 4-wk SARA feeding challenge, although without distinction between responders and nonresponders. At the level of the operational taxonomic unit, we detected diet-specific shifts in epimural community structure, but in the overall epimural bacterial community structure, we found no differences between responders and nonresponders. Correlation analysis revealed significant associations between grain intake and operational taxonomic unit abundance. The study revealed major shifts in the 3 dominating phyla and, most importantly, a loss of diversity in the epimural bacterial communities during a long-term SARA diet challenge, in which 60% concentrate supply for 4 wk was instrumental rather than the magnitude of the drop of ruminal pH below 5.8.

Pyrosequencing reveals shifts in the bacterial epimural community relative to dietary concentrate amount in goats.

Ecological balance in the rumen is highly sensitive to concentrate-rich diets. Yet the effects of these feeding practices on the caprine bacterial epimural microbiome (CBEM), a microbial community with putative important physiological functions in the rumen, are largely unexplored. This study aimed to investigate the effect of dietary concentrate amount on ruminal CBEM. Seventeen growing goats were fed diets with 0 [n=5; 6.2MJ of metabolizable energy (ME)/d], 30 (n=6; 7.3MJ of /d), or 60% (n=6; 10.2MJ of ME/d) concentrate for 6 wk. Two hours after their last feeding, goats were euthanized and tissue samples of the ventral rumen wall were collected, washed in phosphate-buffered saline to detach loosely attached bacteria, and stored at -20°C for further processing. Genomic DNA was isolated from thawed rumen mucosa samples and used for Roche/454 Life Science (Branford, CT) 16S rRNA gene amplicon pyrosequencing yielding 122,458 reads. Pyrosequencing data were clustered into 1,879 operational taxonomic units (OTU; 0.03 distance level). Pyrosequencing revealed Proteobacteria, Bacteroidetes, Firmicutes, and Spirochaetes as the most abundant phyla (97.7%). Compared with the 30% group, both the 60 and 0% concentrate groups harbored significantly more Firmicutes and SR1, respectively. On an OTU level, a Bergeriella-related OTU was most abundant in the CBEM, followed by 2 Campylobacter OTU, which responded differently to diets: 1 OTU was significantly increased whereas the other significantly decreased with highest concentrate amount in the diet. At the genus level, the 0% concentrate group harbored increased Kingella-like sequences compared with the other feeding groups. Furthermore, the 0% concentrate group tended to have more Bergeriella than the 30 and 60% concentrate groups. The genus Bergeriella was significantly decreased in the 60% feeding group compared with the other diets. In conclusion, this is the first report of CBEM using deep-sequencing methods on the genus and OTU level, and our study revealed major shifts in the CBEM in response to concentrate-rich diets with potential health relevance in goats.

Transient feeding of a concentrate-rich diet increases the severity of subacute ruminal acidosis in dairy cattle.

The objective of this study was to investigate the effect of the pattern of concentrate-rich feeding on subacute ruminal acidosis (SARA), its severity, and the corresponding changes in VFA concentration. Eight rumen-cannulated Holstein cows were assigned to a 2 × 2 crossover design with 2 SARA challenge models and 2 experimental runs ( = 8 per treatment). Each run lasted for 40 d, consisting of a 6-d baseline, a 6-d gradual grain adaptation, and a 28-d SARA challenge period. The 2 SARA challenge models were transient (TRA) and persistent (PER) SARA. Initially, all cows were subjected to a forage-only diet (baseline) and gradually switched to 60% concentrate (DM basis). Then, cows in the PER model were continuously challenged for 28 d, whereas cows in the TRA model had a 7-d break from the SARA diet and were fed the forage-only diet after the first 7 d of SARA challenge. Thereafter, the TRA cows were rechallenged with the SARA diet. Wireless ruminal pH sensors were used to obtain ruminal pH profiles and temperature over the experimental period. For the determination of VFA, free ruminal liquid (FRL) and particle-associated ruminal liquid (PARL) were collected once for the baseline and twice (d 20 and 40 for the PER model) or 3 times (d 13, 30, and 40 for the TRA model) during SARA, each time at 0, 4, and 8 h after the morning feeding. Cows in both models experienced SARA albeit with day-to-day variation. From the start until the first 7-d SARA, cows of both models had similar pH profiles, but during the rechallenge, SARA was more severe in the TRA model than in the PER model based on lower daily mean ruminal pH (5.93 vs. 6.15; SEM 0.058) and double the amount of time at pH < 5.8 (497 vs. 278 min; SEM 68.61, < 0.05). Mean ruminal temperature was raised during SARA compared with the baseline (38.9 vs. 38.7°C; SEM 0.057, < 0.001). Concentrations of VFA increased with increasing time after feeding ( < 0.001). In general, SARA challenge (d 40 vs. the baseline), but not the challenge model, altered VFA concentrations and profile of both FRL and PARL by increasing the amounts of propionate and butyrate, whereas total VFA concentration was less affected. Proportions of VFA shifted over the duration of SARA challenge with more propionate but less acetate and butyrate proportions with advancing days of SARA challenge, leading to the values of the last SARA day being different from the earlier days ( < 0.05). In conclusion, the TRA condition led to the higher severity of SARA, but factors beyond feed intake and VFA alterations seemed to play a role.

Technical note: Evaluation of a real-time wireless pH measurement system relative to intraruminal differences of digesta in dairy cattle.

The aim of this study was to evaluate the accuracy and precision of indwelled wireless sensors relative to intrareticuloruminal differences in dairy cows transitioned from a forage to a high-concentrate diet. A feeding trial was performed with 8 rumen-cannulated Holstein cows. The cows were stepwise switched from 0 to 60% concentrate in the diet and fed 5 wk. Samples from the free ruminal liquid (FRL) from the ventral rumen and from the particle-associated ruminal liquid (PARL) in the rumen mat were manually taken at 0, 4, and 8 h after the morning feeding on d 0, 7, 14, and 34 of the experiment through the ruminal cannula to measure pH in FRL and PARL using a pH electrode. Additionally indwelling reticular wireless pH sensors were used to measure reticular pH every 10 min throughout the experiment. Precision and accuracy properties as a measure of reproducibility of the methods were statistically evaluated. Data showed significant differences among pH readings of indwelling sensors and pH measurements taken by means of a conventional electrode in both FRL and PARL (P<0.05). These differences became more evident when 60% concentrate diet was fed. Across all experimental days, the pH of the FRL was greatest and the pH reported by indwelling sensors intermediate, whereas the pH of PARL was lowest. The concordance correlation coefficient (CCC) analysis revealed a high agreement between indwelling sensors and FRL (CCC=0.709) but a low agreement with the pH of PARL (CCC=0.495). In conclusion, the study indicated that wireless sensors can satisfactorily reflect the pH of FRL but poorly reflect that of PARL.