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.

The impact of a long-term subacute rumen acidosis (SARA) on the bovine epimural bacterial microbiome (BEBM) and its consequences for rumen health is poorly understood. This study aimed to investigate shifts in the BEBM during a long-term transient SARA model consisting of two concentrate-diet-induced SARA challenges separated by a 1-week challenge break. Eight cows were fed forage and varying concentrate amounts throughout the experiment. In total, 32 rumen papilla biopsies were taken for DNA isolation (4 sampling time points per cow: at the baseline before concentrate was fed, after the first SARA challenge, after the challenge break, and after the second SARA challenge). Ruminal pH was continuously monitored. The microbiome was determined using Illumina MiSeq sequencing of the 16S rRNA gene (V345 region). In total 1,215,618 sequences were obtained and clustered into 6833 operational taxonomic units (OTUs). Campylobacter and Kingella were the most abundant OTUs (16.5 and 7.1%). According to ruminal pH dynamics, the second challenge was more severe than the first challenge. Species diversity estimates and evenness increased during the challenge break compared to all other sampling time points (P < 0.05). During both SARA challenges, Kingella- and Azoarcus-OTUs decreased (0.5 and 0.4 fold-change) and a dominant Ruminobacter-OTU increased during the challenge break (18.9 fold-change; P < 0.05). qPCR confirmed SARA-related shifts. During the challenge break noticeably more OTUs increased compared to other sampling time points. Our results show that the BEBM re-establishes the baseline conditions slower after a SARA challenge than ruminal pH. Key phylotypes that were reduced during both challenges may help to establish a bacterial fingerprint to facilitate understanding effects of SARA conditions on the BEBM and their consequences for the ruminant host.

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.

Short-chain fatty acids (SCFAs) and lactate are endproducts of rumen fermentation and important energy sources for the host ruminant. Because their rapid accumulation results in ruminal acidosis, enhancement of the absorption of SCFA and lactate across reticuloruminal wall is instrumental in increasing energy supply and preventing ruminal acidosis in cattle. This study investigated whether the reticuloruminal absorption of SCFAs and lactate was altered by different strategies of high concentrate feeding. Eight rumen-cannulated, non-lactating Holstein cows were fed a forage-only diet (baseline) and then gradually adapted over 6 d to a 60% concentrate level. Thereafter, this concentrate-rich diet was fed for 4 wk either continuously (Con; n = 8) or interruptedly (Int; n = 8). Absorption of SCFAs and lactate was determined in vivo from the experimental buffer introduced into the washed reticulorumen. The buffer contained acetate, propionate, butyrate and lactate at a concentration of 60, 30, 10 and 5 mmol/L, respectively and Cr-EDTA as a marker for correcting ruminal water fluxes. The reticuloruminal absorption after 35 and 65 min of buffer incubation was measured at the baseline, after 1 wk of 60% concentrate feeding in the interrupted model (Int-1) and after 4 wk of concentrate feeding in both feeding models (Int-4 and Con-4). Data showed that the absorption rates of individual and total SCFAs during the first 35 min of incubation of Con-4 were highest (~1.7 times compared to baseline), while Int-1 and Int-4 were similar to respective baseline. Lactate was not absorbed during forage-only baseline and 1-wk concentrate feeding, but after 4-wk feeding of concentrates in both models. In conclusion, SCFAs absorption across the reticulorumen of non-lactating cattle was enhanced by the 4-wk continuous concentrate feeding, which seems to be more advantageous in terms of rumen acidosis prevention compared to the interrupted feeding model. The study provides evidence of lactate absorption across the reticulorumen of non-lactating cattle after both continuous and interrupted 4-wk concentrate feeding.

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.