Subacute Ruminal Acidosis Challenge Research Articles

Postbiotics from Saccharomyces cerevisiae fermentation stabilize microbiota in rumen liquid digesta during grain-based subacute ruminal acidosis (SARA) in lactating dairy cows

BackgroundSubacute ruminal acidosis (SARA) is a common metabolic disorder of high yielding dairy cows, and it is associated with dysbiosis of the rumen and gut microbiome and host inflammation. This study evaluated the impact of two postbiotics from Saccharomyces cerevisiae fermentation products (SCFP) on rumen liquid associated microbiota of lactating dairy cows subjected to repeated grain-based SARA challenges. A total of 32 rumen cannulated cows were randomly assigned to 4 treatments from 4 weeks before until 12 weeks after parturition. Treatment groups included a Control diet or diets supplemented with postbiotics (SCFPa, 14 g/d Original XPC; SCFPb-1X, 19 g/d NutriTek; SCFPb-2X, 38 g/d NutriTek, Diamond V, Cedar Rapids, IA, USA). Grain-based SARA challenges were conducted during week 5 (SARA1) and week 8 (SARA2) after parturition by replacing 20% DM of the base total mixed ration (TMR) with pellets containing 50% ground barley and 50% ground wheat. Total DNA from rumen liquid samples was subjected to V3–V4 16S rRNA gene amplicon sequencing. Characteristics of rumen microbiota were compared among treatments and SARA stages.ResultsBoth SARA challenges reduced the diversity and richness of rumen liquid microbiota, altered the overall composition (β-diversity), and its predicted functionality including carbohydrates and amino acids metabolic pathways. The SARA challenges also reduced the number of significant associations among different taxa, number of hub taxa and their composition in the microbial co-occurrence networks. Supplementation with SCFP postbiotics, in particular SCFPb-2X, enhanced the robustness of the rumen microbiota. The SCFP supplemented cows had less fluctuation in relative abundances of community members when exposed to SARA challenges. The SCFP supplementation promoted the populations of lactate utilizing and fibrolytic bacteria, including members of Ruminococcaceae and Lachnospiraceae, and also increased the numbers of hub taxa during non-SARA and SARA stages. Supplementation with SCFPb-2X prevented the fluctuations in the abundances of hub taxa that were positively correlated with the acetate concentration, and α- and β-diversity metrics in rumen liquid digesta.ConclusionsInduction of SARA challenges reduced microbiota richness and diversity and caused fluctuations in major bacterial phyla in rumen liquid microbiota in lactating dairy cows. Supplementation of SCFP postbiotics could attenuate adverse effects of SARA on rumen liquid microbiota.

Postbiotics from Saccharomyces cerevisiae fermentation stabilize rumen solids microbiota and promote microbial network interactions and diversity of hub taxa during grain-based subacute ruminal acidosis (SARA) challenges in lactating dairy cows.

High-yielding dairy cows are commonly fed high-grain rations. However, this can cause subacute ruminal acidosis (SARA), a metabolic disorder in dairy cows that is usually accompanied by dysbiosis of the rumen microbiome. Postbiotics that contain functional metabolites provide a competitive niche for influential members of the rumen microbiome, may stabilize and promote their populations, and, therefore, may attenuate the adverse effects of SARA. This study used a total of 32 rumen-cannulated lactating dairy cows, which were randomly assigned into four treatments: no SCFP (control), 14 g/d Original XPC (SCFPa), 19 g/d NutriTek (SCFPb-1X), and 38 g/d NutriTek (SCFPb-2X) (Diamond V, Cedar Rapids, IA) from 4 weeks before until 12 weeks after parturition. Grain-based SARA challenges were conducted during week 5 (SARA1) and week 8 (SARA2) after parturition by replacing 20% dry matter of the base total mixed ration (TMR) with pellets containing 50% ground barley and 50% ground wheat. The DNA of rumen solids digesta was extracted and subjected to V3-V4 16S rRNA gene sequencing. The characteristics of rumen solids microbiota were compared between non-SARA (Pre-SARA1, week 4; Post-SARA1, week 7; and Post-SARA2, weeks 10 and 12) and SARA stages (SARA1/1, SARA1/2, SARA2/1, SARA2/2), as well as among treatments. Both SARA challenges reduced the richness and diversity of the microbiota and the relative abundances of the phylum Fibrobacteres. Supplementation with SCFP promoted the growth of several fibrolytic bacteria, including Lachnospiraceae UCG-009, Treponema, unclassified Lachnospiraceae, and unclassified Ruminococcaceae during the SARA challenges. These challenges also reduced the positive interactions and the numbers of hub taxa in the microbiota. The SCFPb treatment increased positive interactions among microbial members of the solids digesta and the number of hub taxa during the SARA and non-SARA stages. The SCFPb-2X treatment prevented changes in the network characteristics, including the number of components, clustering coefficient, modularity, positive edge percentage, and edge density of the microbiota during SARA challenges. These challenges reduced predicted carbohydrate and nitrogen metabolism in microbiota, whereas SCFP supplementation attenuated those reductions. Supplementation with SCFP, especially the SCFPb-2X attenuated the adverse effects of grain-based SARA on the diversity and predicted functionality of rumen solids microbiota.

Invited review: Effect of subacute ruminal acidosis on gut health of dairy cows.

Subacute ruminal acidosis (SARA) is assumed to be a common disease in high-yielding dairy cows. Despite this, the epidemiological evidence is limited by the lack of survey data. The prevalence of SARA has mainly been determined by measuring the pH of ruminal fluid collected using rumenocentesis. This may not be sufficiently accurate, because the symptoms of SARA are not solely due to ruminal pH depression, and ruminal pH varies among sites in the rumen, throughout a 24-h period, and among days. The impact of SARA has mainly been studied by conducting SARA challenges in cows, sheep, and goats based on a combination of feed restriction and high-grain feeding. The methodologies of these challenges vary considerably among studies. Variations include differences in the duration and amount of grain feeding, type of grain, amount and duration of feed restriction, number of experimental cows, and sensitivity of cows to SARA challenges. Grain-based SARA challenges affect gut health. These effects include depressing the pH in, and increasing the toxin content of, digesta. They also include altering the taxonomic composition of microbiota, reducing the functionality of the epithelia throughout the gastrointestinal tract (GIT), and a moderate inflammatory response. The effects on the epithelia include a reduction in its barrier function. Effects on microbiota include reductions in their richness and diversity, which may reduce their functionality and reflect dysbiosis. Changes in the taxonomic composition of gut microbiota throughout the GIT are evident at the phylum level, but less evident and more variable at the genus level. Effects at the phylum level include an increase in the Firmicutes to Bacteroidetes ratio. More studies on the effects of a SARA challenge on the functionality of gut microbiota are needed. The inflammatory response resulting from grain-based SARA challenges is innate and moderate and mainly consists of an acute phase response. This response is likely a combination of systemic inflammation and inflammation of the epithelia of the GIT. The systemic inflammation is assumed to be caused by translocation of immunogenic compounds, including bacterial endotoxins and bioamines, through the epithelia into the interior circulation. This translocation is increased by the increase in concentrations of toxins in digesta and a reduction of the barrier function of epithelia. Severe SARA can cause rumenitis, but moderate SARA may activate an immune response in the epithelia of the GIT. Cows grazing highly fermentable pastures with high sugar contents can also have a low ruminal pH indicative of SARA. This is not accompanied by an inflammatory response but may affect milk production and gut microbiota. Grain-based SARA affects several aspects of gut health, but SARA resulting from grazing high-digestible pastures and insufficient coarse fiber less so. We need to determine which method for inducing SARA is the most representative of on-farm conditions.

Saccharomyces cerevisiae fermentation products reduce bacterial endotoxin concentrations and inflammation during grain-based subacute ruminal acidosis in lactating dairy cows

Subacute ruminal acidosis (SARA) is a metabolic disorder in dairy cows that is associated with dysbiosis of rumen and hindgut microbiomes, translocation of immunogenic compounds from the gut lumen into blood circulation, and systemic inflammatory response. In this study we hypothesized that Saccharomyces cerevisiae fermentation products (SCFP) attenuate the increases in ruminal and peripheral bacterial endotoxin concentrations and the inflammation resulting from repeated induction of SARA. Lactating Holstein dairy cows (parity 2 and 3+, n = 32) were fed diets with or without SCFP (all from Diamond V) and subjected to 2 episodes of SARA challenges. Cows received a basal total mixed ration (TMR) containing 34% neutral detergent fiber and 18.6% starch, dry matter (DM) basis. Treatments were randomly assigned to control (basal TMR and 140 g/d of ground corn with no SCFP) or 1 of 3 SCFP treatments: basal TMR and 14 g/d Original XPC (SCFPa), 19 g/d NutriTek (SCFPb-1×), or 38 g/d NutriTek (SCFPb-2×) mixed with 126, 121, or 102 g/d of ground corn, respectively. Treatments were implemented from 4 wk before until 12 wk after parturition. During wk 5 (SARA1) and wk 8 of lactation (SARA2), grain-based SARA challenges were conducted by gradually replacing 20% of DM of the basal TMR over 3 d with pellets containing 50% wheat and 50% barley. Ruminal fluid, fecal, and blood samples were collected weekly during Pre-SARA1 (wk 4, as baseline), Post-SARA1 (wk 7), and Post-SARA2 (wk 10 for blood and wk 12 for rumen and fecal parameters) stages, and twice a week during the challenges SARA1 and SARA2. Rumen papillae samples were taken only during Pre-SARA1 and Post-SARA2. We measured the concentrations of free lipopolysaccharides (LPS) in the rumen fluid and feces; free LPS and lipoteichoic acid (LTA) endotoxins in peripheral plasma; interleukin (IL)-1β and IL-6 in peripheral serum; acute-phase proteins, serum amyloid A (SAA), and LPS-binding protein in peripheral plasma; haptoglobin (Hp) in peripheral serum; and myeloperoxidase (MPO) in rumen papillae. Induction of SARA episodes increased free LPS concentrations in rumen fluid and tended to increase LTA in peripheral plasma. The SARA episodes increased concentration of circulating SAA and tended to increase that of IL-1β compared with Pre-SARA1. Induction of SARA did not affect the concentrations of circulating IL-6, Hp, and MPO. The SCFP supplementation reduced plasma concentrations of LTA and SAA and serum concentration of IL-1β compared with control. Additionally, SCFPb-2× tended to reduce ruminal LPS in second-parity cows compared with control. Overall, SCFP supplementation appeared to stabilize the rumen environment and reduce proinflammatory status, hence attenuating adverse digestive and inflammatory responses associated with SARA episodes.

Long-term effect of subacute ruminal acidosis on the morphology and function of rumen epithelial barrier in lactating goats

Grain-induced subacute ruminal acidosis (SARA) impairs rumen epithelial barrier function, but it is yet to be determined if SARA can cause persistent damage to the morphology and function of the rumen epithelial barrier. The objective of the present study was to investigate if SARA has persistent effects on the morphological structure and permeability of ruminal epithelium and the expression of the genes involved in epithelial barrier function using a lactating goat model. Twelve mid-lactating Saanen goats with rumen cannulas were randomly assigned to 1 of 2 groups: control group (Ctrl, n=4) fed a basal diet with a non-fiber carbohydrate (NFC) to neutral detergent fiber (NDF) ratio of 1.40, and SARA group (SARA, n=8) fed the same basal diet but with increasing NFC to NDF ratio from 1.4 to 1.79, 2.31, and 3.23 overtime to induce SARA. At the end of the SARA challenge (post-SARA), 4 goats were randomly selected from the SARA group and fed only hay mixture adlibitum for another 4 weeks to allow for restitution (post-SARA). Ruminal pH was continuously recorded to monitor the severity of SARA. Samples of the ventral ruminal epithelium were collected after slaughter to examine the structural and functional changes of the ruminal epithelium using transmission electron microscopy (TEM), Ussing chambers, qRT-PCR, and Western bolt analyses. Compared with the Ctrl group, ruminal papilla length, width, surface area and thickness of stratum corneum increased (P<0.05), while stratum spinosum and basale thickness, and total depth of the epithelium decreased (P<0.05) in the SARA group. These changes diminished or tended to return to the levels of the Ctrl group in the post-SARA group (P>0.05). The SARA challenge also decreased cellular junction and widened the intercellular space between epithelial cells. Rumen transepithelial short-circuit current (Isc), tissue conductance (Gt), and mucosa-to-serosa flux of paracellular horseradish peroxidase (HRP) all increased (P<0.05) both in the SARA and post-SARA groups, which indicates that SARA can induce a sustained increase in epithelial permeability and barrier dysfunction. Moreover, the mRNA and protein expressions of CLDN1, OCLN and ZO-1 were down-regulated (P<0.01) in both the SARA and post-SARA groups. The results of this study showed that SARA could result in sustained epithelial barrier dysfunction, at both structural and functional levels, which is associated with decreased expression of rumen epithelial tight junction proteins, and the restitution of rumen epithelial barrier function is slower than that of its morphology.

A Grain-Based SARA Challenge Affects the Composition of Epimural and Mucosa-Associated Bacterial Communities throughout the Digestive Tract of Dairy Cows.

Simple SummaryHigh-yielding dairy cows must receive high-grain diets in order to meet their high energy requirements. However, these diets depress the pH in the rumen, leading to a condition referred to as subacute ruminal acidosis (SARA), and in the large intestine, and may negatively affect the taxonomic composition and the functionality of the populations of microorganisms in the digestive tract. As cows depend on these microorganisms for nutrient utilization and health, disruptions of their composition and functionality can greatly affect the production, health, and welfare of dairy cows. In our study, SARA was induced experimentally by excessive grain feeding. The taxonomic composition of bacterial populations attached to the epithelia of the digestive tract were determined throughout this tract. Our results show that SARA affected the populations of several taxa of bacteria, which suggests that the beneficial effects of these bacteria may be reduced, and that the digestive tract may be at increased risk of invasion by pathogenic microorganisms. The greatest effects of SARA on the taxonomic composition of bacteria on epithelia were in the rumen and large intestine. Their composition on epithelia in the small intestine was also affected, but the affected groups of bacteria differed from those in the rumen and large intestine.The effects of a subacute ruminal acidosis (SARA) challenge on the composition of epimural and mucosa-associated bacterial communities throughout the digestive tract were determined in eight non-lactating Holstein cows. Treatments included feeding a control diet containing 19.6% dry matter (DM) starch and a SARA-challenge diet containing 33.3% DM starch for two days after a 4-day grain step-up. Subsequently, epithelial samples from the rumen and mucosa samples from the duodenum, proximal, middle and distal jejunum, ileum, cecum and colon were collected. Extracted DNA from these samples were analyzed using MiSeq Illumina sequencing of the V4 region of the 16S rRNA gene. Distinct clustering patterns for each diet existed for all sites. The SARA challenge decreased microbial diversity at all sites, with the exception of the middle jejunum. The SARA challenge also affected the relative abundances of several major phyla and genera at all sites but the magnitude of these effects differed among sites. In the rumen and colon, the largest effects were an increase in the relative abundance of Firmicutes and a reduction of Bacteroidetes. In the small intestine, the largest effect was an increase in the relative abundance of Actinobacteria. The grain-based SARA challenge conducted in this study did not only affect the composition and cause dysbiosis of epimural microbiota in the rumen, it also affected the mucosa-associated microbiota in the intestines. To assess the extent of this dysbiosis, its effects on the functionality of these microbiota must be determined in future.

Effect of an intramammary lipopolysaccharide challenge on the hindgut microbial composition and fermentation of dairy cattle experiencing intermittent subacute ruminal acidosis

Feeding grain-rich diets often results in subacute ruminal acidosis (SARA), a condition associated with ruminal dysbiosis and systemic inflammation. Yet, the effect of SARA on hindgut microbiota, and whether this condition is aggravated by exogenous immune stimuli, is less understood. Therefore, the aims of this study were to determine the effects of an intermittent high-grain SARA model on the hindgut microbial community, and to evaluate whether the effects of SARA on the fecal microbiome and fermentation were further affected by an intramammary lipopolysaccharide (LPS) challenge. A total of 18 early-lactating Simmental cows were divided into 3 groups (n = 6); 2 were fed a SARA-inducing feeding regimen (60% concentrate), 1 was fed a control (CON) diet (40% concentrate). On d 30, 1 SARA group (SARA-LPS) and the CON group (CON-LPS) were intramammarily challenged with a single dose of 50 µg of LPS from Escherichia coli O26:B6, whereas the remaining 6 SARA cows (SARA-PLA) received a placebo. Using a longitudinal randomized controlled design, with grouping according to parity and days in milk), statistical analysis was performed with baseline measurements used as a covariate in a mixed model procedure. The SARA-inducing feeding challenge resulted in decreased fecal pH and increased butyrate as a proportion of total short-chain fatty acids in the feces. On d 30, SARA-challenged cows had decreased fecal diversity as shown by the Shannon and Chao1 indices and a decrease in the relative abundance of Euryarchaeota and cellulolytic genera, and numerical increases in the relative abundance of several Firmicutes associated with starch and secondary fermentation. The LPS challenge did not affect the fecal pH and short-chain fatty acids, but increased the Chao1 richness index in an interaction with the SARA challenge, and affected the relative abundance of Verrucomicrobia (1.13%), Actinobacteria (0.19%), and Spirochaetes (0.002%), suggesting an effect on the microbial ecology of the hindgut during SARA conditions. In conclusion, the SARA-inducing feeding regimen promoted important microbial changes at d 30, including reduced diversity and evenness compared with CON, whereas the external LPS challenge led to changes in the microbial community without affecting fecal fermentation properties.

Anti-lipopolysaccharide antibody administration mitigates ruminal lipopolysaccharide release and depression of ruminal pH during subacute ruminal acidosis challenge in Holstein bull cattle.

The effects of anti-lipopolysaccharide (LPS) antibody on rumen fermentation and LPS activity were investigated during subacute ruminal acidosis (SARA) challenge. Eleven Holstein cattle (164 ± 14 kg) were used in a 3 × 3 Latin square design. Cattle were fed a roughage diet on days −11 to −1 (pre-challenge) and day 2 (post-challenge), and a high-grain diet on days 0 and 1 (SARA challenge). For 14 days, 0-, 2-, or 4-g of anti-LPS antibody was administered once daily through a rumen fistula. Ruminal pH was measured continuously, and rumen fluid and blood samples were collected on days −1, 0, 1, and 2. Significantly lower ruminal LPS activity on day 1 was observed in the 2- and 4-g groups than those in the 0-g group. In addition, significantly higher 1-hr mean ruminal pH on SARA challenge period (days 0 and 1) was identified in the 4-g group than in the 0-g group. However, rumen fermentation measurements (total volatile fatty acid [VFA], VFA components, NH3-N and lactic acid) and peripheral blood metabolites (glucose, free fatty acid, beta-hydroxybutyrate, total cholesterol, blood urea nitrogen, aspartate aminotransferase and gamma-glutamyl transferase) were not different among the groups during the experimental periods. Therefore, anti-LPS antibody administration mitigates LPS release and pH depression without the depression of rumen fermentation and peripheral blood metabolites during SARA challenge in Holstein cattle.

Potential protective effects of thiamine supplementation on the ruminal epithelium damage during subacute ruminal acidosis.

In ruminants, the ruminal epithelium not only has the function of absorbing nutrients but also is an important tissue to prevent harmful substances in the rumen from entering the blood circulation. Thus, the normal function of ruminal epithelium is critical for ruminants. However, subacute ruminal acidosis induced by high-concentrate diets often damages the barrier function of ruminal epithelium in ruminants. Recently, many studies have shown that dietary supplementation with thiamine is an effective method to alleviate subacute ruminal acidosis. In order to provide theoretical reference for the in-depth study of subacute ruminal acidosis and the application of thiamine in the future, this review introduces the effects of subacute ruminal acidosis on morphological structure, inflammatory response, and tight junction of ruminal epithelium. In addition, this paper summarizes the role of thiamine in maintaining ruminal epithelial function of ruminants during subacute ruminal acidosis challenge.

Effects of Saccharomyces cerevisiae fermentation products and subacute ruminal acidosis on feed intake, fermentation, and nutrient digestibilities in lactating dairy cows

Effects of Saccharomyces cerevisiae fermentation products (SCFP) and subacute ruminal acidosis (SARA) on rumen and hindgut fermentation, feed intake, and total tract nutrient digestibilities were determined in 32 lactating Holstein cows between weeks 4 and 9 of lactation. Treatments included control, 14 g·d−1 Diamond V Original XPC™ (SCFPa; Diamond V, Cedar Rapids, IA, USA), 19 g·d−1 NutriTek® (SCFPb-1X; Diamond V), and 38 g·d−1 NutriTek® (SCFPb-2X; Diamond V). During weeks 5 and 8, SARA challenges were conducted by switching from a 18.6% to a 27.9% dry matter (DM) starch diet. This reduced the rumen and feces pH. The durations of the rumen pH below 5.6 during these challenges averaged 175.0, 233.8, 246.9, and 79.3 min·d−1 for the control, SCFPa, SCFPb-1X, and SCFPb-2X treatments, respectively. Hence, SARA was not induced under the SCFPb-2X treatment. The feces pH during the SARA challenges was lowest during SCFPb-2X, suggesting this treatment shifted fermentation from the rumen to the hindgut. The SARA challenges reduced the total tract digestibility of DM, neutral detergent fiber digestibility (NDFd), and phosphorus, but tended to increase that of starch. The SCFPb-2X treatment increased the NDFd from 52.7% to 61.8% (P &lt; 0.05). The SCFPb-2X treatment attenuated impacts of SARA.

PacBio and Illumina MiSeq Amplicon Sequencing Confirm Full Recovery of the Bacterial Community After Subacute Ruminal Acidosis Challenge in the RUSITEC System

The impact of subacute rumen acidosis (SARA) on the rumen bacterial community has been frequently studied in in vivo trials. Here we investigated whether these alterations can be mirrored by using the rumen simulation technique (RUSITEC) as an in vitro model for this disease. We hypothezised that the bacterial community fully recovers after a subacute ruminal acidosis challenge. We combined a PacBio nearly full-length 16S rRNA gene analysis with 16S rRNA gene Illumina MiSeq sequencing of the V4 hypervariable region. With this hybrid approach, we aimed to get an increased taxonomic resolution of the most abundant bacterial groups and an overview of the total bacterial diversity. The experiment consisted of a control period I and a SARA challenge and ended after a control period II, of which each period lasted 5 d. Subacute acidosis was induced by applying two buffer solutions, which were reduced in their buffering capacity (SARA buffers) during the SARA challenge. Two control groups were constantly infused with the standard buffer solution. Furthermore, the two SARA buffers were combined with three different feeding variations, which differed in their concentrate-to-hay ratio. The induction of SARA led to a decrease in pH below 5.8, which then turned into a steady-state SARA. Decreasing pH values led to a reduction in bacterial diversity and richness. Moreover, the diversity of solid-associated bacteria was lower for high concentrate groups throughout all experimental periods. Generally, Firmicutes and Bacteroidetes were the predominant phyla in the solid and the liquid phase. During the SARA period, we observed a decrease in fibrolytic bacteria although lactate-producing and -utilizing families increased in certain treatment groups. The genera Lactobacillus and Prevotella dominated during the SARA period. With induction of the second control period, most bacterial groups regained their initial abundance. In conclusion, this in vitro model displayed typical bacterial alterations related to SARA and is capable of recovery from bouts of SARA. Therefore, this model can be used to mimic SARA under laboratory conditions and may contribute to a reduction in animal experiments.

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.

Effects of a Megasphaera elsdenii oral drench on reticulorumen pH dynamics in lactating dairy cows under subacute ruminal acidosis challenge

Subacute ruminal acidosis (SARA) is an important disorder in dairy cattle with economic and animal welfare implications. A preventive strategy for SARA control is oral drenching of probiotics, such as Megasphaera elsdenii. The aim of this study was to evaluate the effects of a M. elsdenii oral drench on reticulorumen pH, milk yield and components, and feeding behavior of lactating cows under a SARA challenge. This study consisted of two crossover trials with 8 cows each to determine the efficacy of drenching live culture of M. elsdenii NCIMB 41125. Experimental period of each trial lasted 8 d with a 4 wk washout interval between periods. The first 3 d of each period were considered baseline days. On day 4 of the period, feed delivered was reduced by half, based upon individual mean dry matter intake (DMI) during the baseline period. On day 5 of the period, all cows received a challenge diet rich in highly fermentable carbohydrates offered to the cows for 2 h to induce SARA. Orts were weighed and replaced with regular diet offered ad libitum. The last 3 d of each experimental period were considered recovery days. The difference between each trial was the administration time of M. elsdenii drench, where in Trial 1 it was delivered 4 d before (PRO-4) and in Trial 2 it was delivered the day before (PRO-1) the SARA challenge. Cows were randomly assigned to either a treatment (PRO-4 or PRO-1) or control (CON-4 or CON-1) drench, and reversed on the subsequent period. During Trial 1, PRO-4 cows produced more milk (P < 0.01), with greater protein percentage (P = 0.03) and lesser fat to protein ratio (P = 0.01) compared with control cows. PRO-4 cows also had greater overall reticulorumen pH and experienced shorter and less intense acidosis (P < 0.05) and greater DMI (P = 0.04) compared with control cows. Feeding behavior was not affected by treatment in Trial 1 (P > 0.10). In Trial 2, only feeding time was affected by the treatment, with PRO-1 cows spending more minutes per day feeding (P < 0.01) compared to control cows. The results indicate potential benefits of a M. elsdenii drench on rumen pH dynamics, acidosis resilience, and possibly milk production and feed intake. However, it appears that the time of drench administration might influence M. elsdenii establishment and drench efficacy.

Potential for a localized immune response by the ruminal epithelium in nonpregnant heifers following a short-term subacute ruminal acidosis challenge

The aim of this study was to investigate whether the ruminal epithelium activates a local inflammatory response following a short-term subacute ruminal acidosis (SARA) challenge. Seven ruminally cannulated, nonpregnant, nonlactating beef heifers, fed a baseline total mixed ration (TMR) with 50:50 forage-to-concentrate ratio, were used in a crossover design with 2 periods and 2 treatments: SARA and control (CON). Induction of SARA included feed restriction (25% of dry matter intake [DMI] for 24 h) followed by a grain overload (30% of baseline DMI) and provision of the full TMR; whereas, the CON group received the TMR ad libitum. Ruminal pH was recorded using indwelling probes, and ruminal lipopolysaccharide (LPS) concentration was measured daily following the challenge until d 6. Biopsies of ruminal papillae from the ventral sac were collected on d 2 and 6 after the grain overload. Transcript abundance of genes associated with acute inflammation was measured by quantitative real-time PCR, normalized to the geometric mean of 3 stable housekeeping genes. Target genes included toll-like receptor-2 (TLR2), TLR4, TLR9, tumor necrosis factor-α (TNFA), prostaglandin endoperoxide synthase-1 (PTGS1), PTGS2 transforming growth factor β-1 (TGFB1), and 4 intermediate enzymes of leukotriene synthesis (ALOX5, ALOX5AP, LTA4H, and LTC4S). Protein localization and expression of TLR4 were quantified by image analysis of fluorescence intensity. Statistical analysis was performed using as a crossover design with fixed effects of treatment, day, and the treatment × day interaction with the random effect of day within period. Ruminal pH was below 5.6 for 4.5 h/d and below 5.8 for 6.9 h/d in the SARA group compared with 22 and 72 min/d, respectively, for CON. Ruminal LPS concentration peaked on d 2 in SARA heifers at 51,481 endotoxin units (EU)/mL compared with 13,331 EU/mL in CON. Following grain overload, small but statistically significant decreases in the transcriptional abundance of TLR2, TLR4, TNF, PTGS2, ALOX5, and ALOX5AP were seen in SARA versus CON heifers. A functionally relevant decrease in TLR4 expression in SARA heifers compared with CON was confirmed by a decrease in fluorescence intensity of the corresponding protein following immunohistofluorescent staining of papillae. The study results indicate a suppression of the inflammatory response in the ruminal epithelium and suggest that the response is tightly regulated, allowing for tissue recovery and return to homeostasis following SARA.

Susceptibility of dairy cows to subacute ruminal acidosis is reflected in milk fatty acid proportions, with C18:1 trans-10 as primary and C15:0 and C18:1 trans-11 as secondary indicators

The current study was carried out to assess 2 hypotheses: (1) cows differ in susceptibility to a subacute ruminal acidosis (SARA) challenge, and (2) the milk fatty acid (FA) pattern can be used to differentiate susceptible from nonsusceptible cows. For this, 2 consecutive experiments were performed. During experiment 1, the milk FA pattern was determined on 125 cows fed an increasing amount of concentrate during the first 4 wk in milk (WIM). The coefficient of variation of several SARA indicative milk FA (i.e., C15:0, C18:1 trans-10, C18:2 cis-9,trans-11, and C18:1 trans-10 to C18:1 trans-11 ratio) increased, indicating that cows reacted differently upon the concentrate build-up. A first grouping was based on the milk fat C18:1 trans-10 proportion in the third WIM. Fifteen cows with the highest proportion of the latter FA (HT10) and their counterparts with low C18:1 trans-10 and equal parity distribution (LT10) were compared, which revealed that milk fat content and milk fat to protein ratio were lower for the HT10 group. From each of the HT10 and LT10 groups, 5 animals were selected for experiment 2. The subselection of the HT10 group, referred to as HT10s, showed a high proportion of C18:1 trans-10 at 3 WIM (>0.31 g/100 g of FA), a high level of C15:0 (on average ≥1.18 g/100 g of FA over the 4 WIM), and a sharp decrease of C18:1 trans-11 (Δ ≥ 0.25 g/100 g of FA during the 4 WIM). Their counterparts (LT10s) had a low milk fat C18:1 trans-10 proportion at 3 WIM (<0.23 g/100 g of FA), an average C15:0 proportion of 0.99 g/100 g of FA or lower, and a rather stable C18:1 trans-11 proportion. The HT10s group was hypothesized to be more susceptible to a SARA challenge, achieved by increasing amounts of rapidly fermentable carbohydrates in experiment 2. The HT10s cows had a lower nadir, mean, and maximum reticulo-ruminal pH; longer period of reticulo-ruminal pH below 6.0; and higher daily reticulo-ruminal pH variation compared with LT10s cows. Throughout experiment 2, HT10s and LT10s cows differed in levels of SARA indicative milk FA. Five animals, including one LT10s and 4 HT10s cows, experienced SARA, defined as reticulo-ruminal pH <6.0 for more than 360 min/d. These results indicate that it is possible to distinguish cows with different susceptibility to a SARA challenge within a herd by monitoring the milk FA composition when cows receive the same diet.

Supplementing phytogenic compounds or autolyzed yeast modulates ruminal biogenic amines and plasma metabolome in dry cows experiencing subacute ruminal acidosis

Subacute ruminal acidosis (SARA) causes ruminal dysbiosis, thereby increasing the risk of systemic metabolic disorders in cattle. We recently showed that supplementation with phytogenic compounds (PHY) or autolyzed yeast (AY) counteracted negative effects of SARA by improving ruminal pH and microbiome. This study investigated the effects of an intermittent SARA challenge on the ruminal concentration of biogenic amines (BA) and lipopolysaccharides (LPS), as well as on the blood metabolome. We also evaluated effects of PHY and AY on the latter variables. Eight rumen-cannulated nonlactating Holstein cows were arranged in an incomplete 4 × 3 Latin square design with 4 experimental runs and 3 treatment groups. During each run, cows were switched from an all-forage diet (baseline) to an intermittent concentrate-challenge diet with a forage:concentrate ratio of 35:65 (dry matter basis) to induce SARA for 1 (SARA1) or 2 (SARA2) wk, separated by 1 wk of forage-only feeding. The 3 treatment groups were no additive as control, PHY, or AY. During baseline, SARA1 and SARA2 rumen fluid samples were collected for analysis of BA and LPS. Blood samples were taken during baseline and SARA1 for a targeted metabolomics approach. High-concentrate feeding caused a 9-fold increase in ruminal LPS during SARA1 and an 11-fold increase in SARA2 compared with the baseline. Elevated concentrations of ruminal BA were found during both SARA periods, with histamine showing the strongest increase during SARA1. Moreover, a decrease in phosphatidylcholines, lysophosphatidylcholines, sphingomyelines, and several AA in the blood during SARA1 were detected. Supplementation of PHY decreased concentrations of LPS (-43%), histamine (-66%), pyrrolidine (-38%), and spermine (-54%) in SARA1 and cadaverine in SARA2 (-50%). Moreover, cows that received PHY had higher concentrations of cholesterol (+26%), several AA, and phosphatidylcholines in SARA1 compared with control cows. For AY, decreases in ruminal ethanolamine (-21%), methylamine (-52%), histamine (-54%), spermidine (-44%), and spermine (-80%) in SARA1 were observed, whereas in the blood an increase in tryptophan was noticed. In conclusion, the SARA was associated with markedly increased concentrations of LPS and BA in the rumen fluid and undesirable shifts in the plasma metabolome. Supplementation of PHY and AY counteracted some of these changes and therefore may help in attenuating negative effects of high-concentrate feeding in dairy cattle.

Effects of repeated subacute ruminal acidosis challenges on the adaptation of the rumen bacterial community in Holstein bulls

We investigated the effect of repeated subacute ruminal acidosis (SARA) challenges on the pH, fermentative function, and bacterial community in the rumen. Four rumen-cannulated Holstein bulls were fed a high-forage diet for 7 d (HF period) followed by a high-grain diet for 7 d (HG period). Four SARA challenges were carried out consecutively (first, second, third, and fourth challenges). The ruminal pH was measured continuously during the experiment, and rumen fluid samples during the first to fourth challenges were collected at 0800, 1400, and 2000 h on the last days of each feeding period for analysis; volatile fatty acid components, NH3-N, and lactic acid concentrations were measured. Bacterial community structure was analyzed at 0800 h during the first and fourth challenges on the last days of each period. The 24-h mean ruminal pH was decreased during the transition from high-forage to high-grain diet and tended to differ between the HF and HG periods. During the HG period, ruminal pH <5.6 was maintained for a longer period in the first and second challenges (350 and 405 min/d, respectively) than in the third and fourth challenges (both 120 min/d). A marked increase in total volatile fatty acid and NH3-N concentrations during the HG period was observed in the later challenges. In addition, lower and higher proportions of acetic and butyric acids, respectively, were observed during the HG period than during the HF period. A total of 37 core bacterial genera were found in all samples; however, the relative abundance of several genera differed significantly between the HF and HG periods (Prevotella, Ruminococcus, Eubacterium, and Oscillibacter) and between the first and fourth challenges (Eubacterium and unclassified Clostridiaceae). During the HG period, lower relative abundances of Prevotella, Eubacterium, and Oscillibacter and higher relative abundance of Ruminococcus were detected compared with during the HF period. The relative abundances of Eubacterium and unclassified Clostridiaceae were lower in the first challenge than in the fourth challenge. Bacterial diversity was greater during the HF period than during the HG period and was greater during the fourth challenge than during the first challenge. Interestingly, diversity indices during the HG period of the fourth challenge were higher than those during the HF period of the first challenge. These results suggested that rumen fermentation in Holstein cattle can adapt to repeated SARA challenges by minimizing the adverse changes in ruminal pH. Moreover, the composition and diversity of the ruminal bacterial community may be affected by ruminal pH and vice versa.

Changes in Microbiota in Rumen Digesta and Feces Due to a Grain-Based Subacute Ruminal Acidosis (SARA) Challenge.

The effects of a grain-based subacute ruminal acidosis (SARA) challenge on bacteria in the rumen and feces of lactating dairy cows were determined. Six lactating, rumen-cannulated Danish Holstein cows were used in a cross-over study with two periods. Periods included two cows on a control diet and two cows on a SARA challenge. The control diet was a total mixed ration containing 45.5% dry matter (DM), 43.8% DM neutral detergent fiber, and 19.6% DM starch. The SARA challenge was conducted by gradually substituting the control diet with pellets containing 50% wheat and 50% barley over 3days to reach a diet containing 55.6% DM, 31.3% DM neutral detergent fiber, and 31.8% DM starch, which was fed for four more days. Rumen fluid samples were collected at day 7 and 10 of experimental periods. Feces samples were collected on days 8 and 10 of these periods. Extracted DNA from the rumen and feces samples was analyzed to assess their bacterial communities using MiSeq Illumina sequencing of the V4 region of the 16S rRNA gene. The induction of SARA reduced the richness, diversity, and stability of bacterial communities and resulted in distinctly different microbiota in the rumen and feces. Bacteroidetes and Firmicutes were the most abundant phyla and, combined, they represented 76.9 and 94.4% of the bacterial community in the rumen fluid and the feces, respectively. Only the relative abundance of Firmicutes in the rumen was increased by the SARA challenge. In rumen fluid and feces, the abundances of nine out of the 90 and 25 out of the 89 taxa, respectively, were affected by the challenge. Hence, SARAchallenge altered the composition of the bacterial community at the lower taxonomical level in the feces and therefore also likely in the hindgut, as well as in the rumen. However, only reductions in the bacterial richness and diversity in the rumen fluid and feces were in agreement with those of other studies and had a biological basis. Although the composition of the bacterial community of the feces was affected by the SARA challenge, bacterial taxa in the feces that can be used for accurate and non-invasive diagnosis of SARA could not be identified.

Nutritional Models of Experimentally-Induced Subacute Ruminal Acidosis (SARA) Differ in Their Impact on Rumen and Hindgut Bacterial Communities in Dairy Cows.

Effects of subacute ruminal acidosis (SARA) challenges on the bacteria in rumen fluid, cecal digesta, and feces of dairy cows were determined using 16S rRNA gene pyrosequencing and real-time quantitative PCR. Six non-lactating Holstein cows with cannulas in the rumen and cecum were used in a 3 × 3 Latin square arrangement of treatments. During the first 3 wk of each experimental period, cows received a control diet containing 70% forages on a dry matter (DM) basis. In wk 4 of each period, cows received one of three diets: (1) the control diet; (2) a diet in which 34% of the dietary DM was replaced with pellets of ground wheat and barley (GBSC); or (3) a diet in which 37% of dietary DM was replaced with pellets of ground alfalfa (APSC). Rumen fluid, cecal digesta and feces were collected on d 5 of wk 4 of each period and the composition of the bacterial community was studied. Rumen fermentation responses were reported in a companion study. Both SARA-inducing challenges resulted in similar digesta pH depressions (as shown by the companion study), and reduced bacterial richness and diversity in rumen fluid, but GBSC had the larger effect. None of the challenges affected these measures in cecal digesta, and only GBSC reduced bacterial richness and diversity in feces. Only GBSC reduced the abundance of Bacteroidetes in rumen fluid. Abundances of limited number of bacterial genera identified by 16S rRNA gene sequencing in the rumen, cecum and feces were affected by the GBSC. The APSC did not affect any of these abundances. Both challenges increased the abundances of several starch, pectin, xylan, dextrin, lactate, succinate, and sugar fermenting bacterial species in the rumen, cecum, and feces as determined by qPCR. Only GBSC increased that of Megasphaera elsdenii in the rumen. Both challenges decreased the abundance of Streptococcus bovis, and increased that of Escherichia coli, in cecal digesta and feces, with GBSC having the larger effect. These results showed that the SARA challenges caused moderate and reversible changes of the composition of the bacteria in the foregut and hindgut, with the greater changes observed during GBSC.

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.