Subacute Ruminal Acidosis Induction Research Articles

Changes in Rumen Epithelial Morphology and Transcriptome, Rumen Metabolome, and Blood Biochemical Parameters in Lactating Dairy Cows with Subacute Rumen Acidosis Following Rumen Content Transplantation

Interventions targeting the gut microbiota, such as fecal microbiota transplantation, prove effective in repairing the intestinal barrier and facilitating the recovery of its function and metabolism. However, the regulatory mechanisms governing the remodeling of rumen epithelial morphology and function, rumen metabolism, and host metabolism in cows of subacute ruminal acidosis (SARA) remain poorly understood. Here, we explored the changes in rumen epithelial morphology and transcriptome, rumen metabolome, and blood biochemical parameters in SARA cows following rumen content transplantation (RCT). The entire experiment consisted of 2 periods: the SARA induction period and the RCT period. During the SARA induction period, 12 ruminally cannulated lactating Holstein cows were randomly allocated into 2 groups, fed either a conventional diet [CON; n = 4; 40% concentrate, dry matter (DM) basis] or a high-grain diet (HG; n = 8; 60% concentrate, DM basis). Following the SARA induction period, the RCT period started. The HG cows were randomly assigned to 2 groups: the donor-recipient (DR) group and the self-recipient (SR) group. Rumen contents were entirely removed from both groups before RCT. For the DR group, cows were administered 70% rumen content from the CON cows, paired based on comparable body weight; for the SR group, each cow received 70% self-derived rumen content. The results revealed no significant differences in the thicknesses of the stratum corneum, granulosum, and spinosum/basale layers, as well as the total depth of the epithelium between the SR and DR groups. All these measurements exhibited a decreasing trend and fluctuations over time after the transfer. Notably, these fluctuations tended to stabilize at 13 or 16 d after RCT in the SR group, whereas they tended to stabilize after 8 or 13 d of transfer for the DR group. Transcriptome sequencing revealed that a total of 277 differentially expressed genes (DEGs) were identified between the 2 groups. Enrichment analysis showed that the DEGs were significantly enriched in 11 Gene Ontology biological processes and 14 KEGG pathways. The DEGs corresponding to almost any of these 11 biological process terms and 14 pathways showed mixed up- or downregulation following RCT. Metabolomics analysis indicated that a total of 33 differential metabolites were detected between the SR and DR groups, mainly enriched in 5 key metabolic pathways, including plant polysaccharides and starch degradation, lipid metabolism, amino sugar and nucleotide metabolism, purine metabolism, and Krebs cycle. Among them, the levels of differential metabolites associated with the degradation of plant polysaccharides and starches, metabolism of amino sugars and nucleotides, and purine metabolism pathways were significantly elevated in the DR cows. The results of blood biochemical parameters showed that the triglyceride concentration of the DR cows was increased than that of the SR cows, comparable to the level observed in the CON cows during the SARA induction period. Generally, our findings indicated that RCT facilitated the recovery of rumen epithelial morphological structure but did not promote its function recovery. Moreover, RCT enhanced rumen plant polysaccharide and starch degradation, amino sugar and nucleotide sugar metabolism, as well as purine metabolism. Additionally, it further promoted the recovery of plasma metabolites related to lipid metabolism.

Sialic acid exacerbates gut dysbiosis-associated mastitis through the microbiota-gut-mammary axis by fueling gut microbiota disruption

BackgroundMastitis is one of the most severe diseases in humans and animals, especially on dairy farms. Mounting evidence indicates that gastrointestinal dysbiosis caused by induction of subacute ruminal acidosis (SARA) by high-grain diet consumption and low in dietary fiber is associated with mastitis initiation and development, however, the underlying mechanism remains unknown.ResultsIn the present study, we found that cows with SARA-associated mastitis have altered metabolic profiles in the rumen, with increased sialic acids level in particular. Consumption of sialic acid (SA) in antibiotic-treated mice, but not healthy mice, induced marked mastitis. SA treatment of antibiotic-treated mice also induced mucosal and systemic inflammatory responses, as evidenced by increased colon and liver injuries and several inflammatory markers. In addition, gut dysbiosis caused by antibiotic impaired gut barrier integrity, which was aggravated by SA treatment. SA potentiated serum LPS level caused by antibiotic treatment, leading to increased activation of the TLR4-NF-κB/NLRP3 pathways in the mammary gland and colon. Moreover, SA facilitated gut dysbiosis caused by antibiotic, and especially enhanced Enterobacteriaceae and Akkermansiaceae, which correlated with mastitis parameters. Fecal microbiota transplantation from SA-antibiotic-treated mice mimicked mastitis in recipient mice. In vitro experiments showed that SA prompted Escherichia coli growth and virulence gene expression, leading to higher proinflammatory cytokine production in macrophages. Targeting the inhibition of Enterobacteriaceae by sodium tungstate or treating with the commensal Lactobacillus reuteri alleviated SA-facilitated mastitis. In addition, SARA cows had distinct ruminal microbial structure by the enrichment of SA-utilizing opportunistic pathogenic Moraxellaceae and the depletion of SA-utilizing commensal Prevotellaceae. Treating mice with the specific sialidase inhibitor zanamivir reduced SA production and Moraxellaceae abundance, and improved mastitis in mice caused by ruminal microbiota transplantation from cows with SARA-associated mastitis.ConclusionsThis study, for the first time, indicates that SA aggravates gut dysbiosis-induced mastitis by promoting gut microbiota disturbance and is regulated by commensal bacteria, indicating the important role of the microbiota-gut-mammary axis in mastitis pathogenesis and suggesting a potential strategy for mastitis intervention based on gut metabolism regulation.-WPXBcu1emhBxPP-wo4QqAVideo

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.

Changes in rumen fermentation and bacterial community in lactating dairy cows with subacute rumen acidosis following rumen content transplantation

Rumen microbiota intervention has long been used to cure ruminal indigestion in production and has recently become a research hotspot. However, how it controls the remodeling of rumen bacterial homeostasis and the restoration of rumen fermentation in cows of subacute ruminal acidosis (SARA) remains poorly understood. This study explored changes in rumen fermentation and bacterial communities in SARA cows following rumen content transplantation (RCT). The entire experiment comprised 2 periods: the SARA induction period and the RCT period. During the SARA induction period, 12 ruminally cannulated lactating Holstein cows were selected and allocated into 2 groups at random, fed either a conventional diet [CON; n = 4; 40% concentrate, dry matter (DM) basis] or a high-grain diet (HG; n = 8; 60% concentrate, DM basis). After the SARA induction period, the RCT period began. The HG cows were randomly divided into 2 groups: the donor-recipient (DR) group and the self-recipient (SR) group, and their rumen contents were removed completely before RCT. For the DR group, cows received 70% rumen content from the CON cows, paired based on comparable body weight; for the SR group, each cow received 70% rumen content, self-derived. The results showed that nearly all rumen fermentation parameters returned to the normal levels that the cows had before SARA induction after 6 d of transplantation, regardless of RCT. The concentrations of acetate, valerate, and total volatile fatty acids (VFA) were not recovered in the SR cows, whereas all of them were recovered in the DR cows. The amplicon sequencing results indicated that both the SR and DR cows rebuild their rumen bacterial homeostasis quickly within 4 d after RCT, and the DR group showed a higher level of bacterial community diversity. At the genus level, the DR cows displayed an improved proportion of unclassified Ruminococcaceae and Saccharofermentans compared with the SR cows. Correlation analysis between the rumen bacteria and rumen fermentation suggested some potential relationships between the predominant transplantation-sensitive operational taxonomic units and VFA. Co-occurrence network analysis revealed that RCT affected only those rumen bacterial taxa that showed weak interactions with other taxa and did not affect the pivotal rumen bacteria with high levels of co-occurrence. Our findings indicate that RCT contributes to the restoration of rumen bacterial homeostasis and rumen fermentation in cows suffering from SARA without affecting the core microbiome.

Subacute Ruminal Acidosis in Zebu Cattle: Clinical and Behavioral Aspects

Simple SummaryCattle that are fed high levels of concentrates may develop short-term rumen acidity that may occur frequently leading to necrosis of the rumen wall and reduced nutrient absorption, thereby decreasing animal productivity. This condition is known as subacute acidosis. Here, we evaluated an experimental model to induce such a condition in Nelore cattle, a Zebu breed widely used in Brazil, and assessed several clinical and feeding behavioral patterns of affected animals to better understand the disease pathogenesis and clinical outcomes. Subacute acidosis led to a reduction in food consumption and rumination time, and an increase was observed in the time spent in decubitus. Additionally, subacute acidosis caused different degrees of depression that was more pronounced with higher ruminal lactic acid concentrations.We evaluated the clinical aspects and feeding behavior of cattle with subacute ruminal acidosis (SARA) caused by short-chain fatty acids (SCFAs). Ten healthy Nelore heifers were subjected to an adjusted SARA induction protocol using citrus pulp (CP). Clinical examinations were performed at baseline and at 3, 6, 9, 12, 15, 18, and 24 h intervals after induction, with ruminal fluid, blood, and feces sampling. The animals’ feeding behavior was evaluated on, before, and for 3 days after SARA by observing the animals every 5 min for 24 h. The dry matter intake (DMI) was recorded daily. The ruminal pH during SARA was always lower than baseline, with an acidotic duration of 547 ± 215 min, a minimum pH of 5.38 ± 0.16, and an average pH of 5.62 ± 0.1. SARA was mainly caused by SCFAs (maximum 118.4 ± 9.3 mmol/L), with the production of l-lactic acids (7.17 mmol/L) and d-lactic acids (0.56 mmol/L) 6 h after the experiment began. The DMI was reduced by 66% and 48% on days 1 and 2, respectively, and returned to normal levels on day 3. SARA caused a reduction in feed intake and rumination time, as well as an increase in the time spent in decubitus on days 1 and 2. These results were influenced by the ruminal pH, ruminal movement, and osmolarity. Furthermore, SARA caused different degrees of depression, which became more pronounced with higher ruminal lactic acid concentrations.

Transient reductions in milk fat synthesis and their association with the ruminal and metabolic profile in dairy cows fed high-starch, low-fat diets

Sub-acute ruminal acidosis (SARA) is sometimes observed along with reduced milk fat synthesis. Inconsistent responses may be explained by dietary fat levels. Twelve ruminally cannulated cows were used in a Latin square design investigating the timing of metabolic and milk fat changes during Induction and Recovery from SARA by altering starch levels in low-fat diets. Treatments were (1) SARA Induction, (2) Recovery and (3) Control. Sub-acute ruminal acidosis was induced by feeding a diet containing 29.4% starch, 24.0% NDF and 2.8% fatty acids (FAs), whereas the Recovery and Control diets contained 19.9% starch, 31.0% NDF and 2.6% FA. Relative to Control, DM intake (DMI) and milk yield were higher in SARA from days 14 to 21 and from days 10 to 21, respectively (P < 0.05). Milk fat content was reduced from days 3 to 14 in SARA (P < 0.05) compared with Control, while greater protein and lactose contents were observed from days 14 to 21 and 3 to 21, respectively (P < 0.05). Milk fat yield was reduced by SARA on day 3 (P < 0.05), whereas both protein and lactose yields were higher on days 14 and 21 (P < 0.05). The ruminal acetate-to-propionate ratio was lower, and the concentrations of propionate and lactate were higher in the SARA treatment compared with Control on day 21 (P < 0.05). Plasma insulin increased during SARA, whereas plasma non-esterified fatty acids and milk β-hydroxybutyrate decreased (P < 0.05). Similarly to fat yield, the yield of milk preformed FA (>16C) was lower on day 3 (P < 0.05) and tended to be lower on day 7 in SARA cows (P < 0.10), whereas yield of de novo FA (<16C) was higher on day 21 (P < 0.01) in the SARA group relative to Control. The t10- to t11-18:1 ratio increased during the SARA Induction period (P < 0.05), but the concentration of t10-18:1 remained below 0.5% of milk fat, and t10,c12 conjugated linoleic acid remained below detection levels. Odd-chain FA increased, whereas branched-chain FA was reduced during SARA Induction from days 3 to 21 (P < 0.05). Sub-acute ruminal acidosis reduced milk fat synthesis transiently. Such reduction was not associated with ruminal biohydrogenation intermediates but rather with a transient reduction in supply of preformed FA. Subsequent rescue of milk fat synthesis may be associated with higher availability of substrates due to increased DMI during SARA.

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.

Induction of Subacute Ruminal Acidosis Affects the Ruminal Microbiome and Epithelium.

Subacute ruminal acidosis (SARA) negatively impacts the dairy industry by decreasing dry matter intake, milk production, profitability, and increasing culling rate and death loss. Six ruminally cannulated, lactating Holstein cows were used in a replicated incomplete Latin square design to determine the effects of SARA induction on the ruminal microbiome and epithelium. Experimental periods were 10 days with days 1–3 for ad libitum intake of control diet, followed by 50% feed restriction on day 4, and ad libitum access on day 5 to the basal diet or the basal diet with an additional 10% of a 50:50 wheat/barley pellet. Based on subsequent ruminal pH, cows were grouped (SARA grouping; SG) as Non-SARA or SARA based on time <5.6 pH (0 and 3.4 h, respectively). Ruminal samples were collected on days 1 and 6 of each period prior to feeding and separated into liquid and solid fractions. Microbial DNA was extracted for bacterial analysis using 16S rRNA gene paired-end sequencing on the MiSeq Illumina platform and quantitative PCR (qPCR). Ruminal epithelium biopsies were taken on days 1 and 6 before feeding. Quantitative RT-PCR was used to determine gene expression in rumen epithelium. Bray–Curtis similarity indicated samples within the liquid fraction separated by day and coincided with an increased relative abundance of genera Prevotella, Ruminococcus, Streptococcus, and Lactobacillus on day 6 (P < 0.06). Although Firmicutes was the predominant phyla in the solid fraction, a SG × day interaction (P < 0.01) indicated a decrease on day 6 for SARA cows. In contrast, phylum Bacteroidetes increased on day 6 (P < 0.01) for SARA cows driven by greater genera Prevotella and YRC22 (P < 0.01). Streptococcus bovis and Succinivibrio dextrinosolvens populations tended to increase on day 6 but were not affected by SG. In ruminal epithelium, CLDN1 and CLDN4 expression increased on day 6 (P < 0.03) 24 h after SARA induction and a tendency for a SG × day interaction (P < 0.10) was observed for CLDN4. Overall, results indicate more rapid adaptation to an induced bout of SARA in the solid fraction ruminal microbiome compared with ruminal epithelium.

Impact of Saccharomyces cerevisiae fermentation product and subacute ruminal acidosis on production, inflammation, and fermentation in the rumen and hindgut of dairy cows

The effects of a Saccharomyces cerevisiae fermentation product (SCFP) on microbial fermentation in the digestive tract, milk production, and inflammation response were determined in Holstein dairy cows under normal and subacute ruminal acidosis (SARA) conditions. Eight lactating dairy cows with ruminal and cecal cannulas were used in a cross-over design with 5 wk experimental periods. During the first experimental period, cows were randomly assigned to treatment, i.e. SCFP (Original XPC, Diamond V, Cedar Rapids, IA) or control. During the second experimental period, treatments were reversed. Experimental periods were separated by a two-week wash out period. During each period, the 4 cows on the SCFP treatment were supplemented with 14g/d of SCFP in 126g of ground corn, whereas the other four cows received 140g ground corn only. During the first 4 wk of each experimental period, all cows received a basal diet. During week 5 of both experimental periods, a SARA challenge was conducted in all cows by replacing 208g/kg of the basal diet with pellets of ground wheat and barley (50:50 on a weight basis). This SARA challenge increased the duration of rumen pH below 5.6 from 11.1 to 311.1min/d, indicating successful induction of SARA. Inducing SARA reduced milk fat from 3.24 to 2.82%, and lowered the acetate to propionate ratio from 3.07 to 1.74 in rumen fluid, from 5.13 to 4.57 in cecal digesta, and from 4.81 to 4.34 in feces. Induction of SARA increased milk protein content from 3.23 to 3.32%, without a concomitant effect on milk protein yield. Inducing SARA also increased endotoxic lipopolysaccharide (LPS) in rumen fluid, cecal digesta and feces from 15,389 to 123,296 endotoxin units (EU)/mL, from 31,982 to 77,380EU/mL, and from 29,679 to 77, 371EU/mL, respectively. Concentrations of LPS, serum amyloid A, haptoglobin, and LPS-binding protein increased by 215.0, 250.6, 181.6 and 47.5%, respectively, in peripheral blood when SARA was induced. The SCFP did not affect feed intake, yields of milk, milk fat, and milk protein but it reduced the variation in rumen pH during control feeding. During the SARA challenge, SCFP tended to reduce ruminal LPS at 6h after feed delivery, increased milk fat percentage (2.71 vs 2.92%, control vs SCFP), and reduced the extent of SARA-associated inflammation response as indicated by a reduction in the concentration of the pro-inflammatory cytokine tumor necrosis factor α (0.09 vs 0.03ng/mL, control vs SCFP). This shows that SCFP can attenuate some of the impact of SARA on production and health of dairy cows.

O uso de monensina sódica e probióticos para o controle de acidose ruminal subaguda em ovinos

The aim of this work was to validate a protocol for induction of subacute ruminal acidosis (SARA) (Experiment 1) and test the efficiency of probiotic Saccharomyces cerevisiae or monensin to avoid pH ruminal drops in sheep (Experiment 2). In Experiment 1, six ewes were fasted for two days and then fed most with concentrate during four days. Ewes in this protocol had ruminal fluid pH below 6.0 and kept it for 75 consecutive hours. In Experiment 2, 18 sheep were distributed into three groups: Control (CG, n = 6), monensin (MG, n = 6) and probiotic group (PG, n = 6). SARA was induced according Experiment 1. PG had lower pH (5.7 ± 0.1) than CG (6.0 ± 0.1) (P = 0.05), while MG (5.7 ± 0.1) was similar to both during SARA induction. SARA induction reduced ruminal protozoa population (P < 0.05) and increased chloride concentrations in ruminal fluid (P < 0.01). In serum, SARA increased concentrations of phosphorus (P < 0.01), AST (P < 0.01) and GGT (P < 0.01), but reduced LDH (P < 0.01). In conclusion, the protocol used for SARA induction was able to maintain ruminal pH between 5.5-6.0 for more than 48 hours. However, monensin and probiotics supplementation was not effective in preventing changes in ruminal and serum parameters during SARA.

Evaluation of the systemic innate immune response and metabolic alterations of nonlactating cows with diet-induced subacute ruminal acidosis

Subacute ruminal acidosis (SARA) increases lipopolysaccharide endotoxin in the rumen, which might translocate into the systemic circulation, triggering a cascade of clinical and immunological alterations. The objective of this study was to characterize the clinical immune and metabolic responses to ruminal-derived lipopolysaccharide in nonlactating cows induced with SARA using 2 challenges, a grain-based SARA challenge (GBSC) or an alfalfa-pellet SARA challenge (APSC). Six dry, nonlactating Holstein cows were used in a 3×3 Latin square arrangement of treatments with 4-wk experimental cycles. All cows received the control diet containing 70% forage and 30% mixed concentrates (dry matter basis) for 3 wk. In wk 4, cows received a control diet, GBSC (38% wheat-barley pellets, 32% other mixed concentrate, and 30% forages), or APSC (45% mixed concentrate, 32% alfalfa pellets, and 23% other forages). Total plasma proteins and immunology-related proteins, acute phase proteins, blood cells, serum chemistry, mRNA gene expression of peripheral blood cell surface markers, and selected proinflammatory cytokines were evaluated. Ruminal pH was lower in both groups with induced SARA compared with a control group. Ruminal endotoxins were higher in GBSC; however, plasma endotoxin was not detected in any study group. No significant differences in feed intake, rectal temperature, white blood cell counts, or differentials were found between control and SARA challenge groups; changes in glucose, urea, Ca, and Mg were observed in SARA groups. Total plasma proteins were lower in both SARA groups, and acute phase proteins were higher in GBSC. The expression of CD14, MD2, and TLR4 mRNA in peripheral blood leukocytes was not affected by SARA induction. The induction of SARA as a result of GBSC or APSC challenge was successful; however, LPS was not detected in plasma. Changes in clinical, metabolic, and inflammatory responses were not observed in the SARA-challenged cows, suggesting that, in this study, SARA was not associated with a systemic response to inflammation.

Impact of subacute ruminal acidosis (SARA) adaptation on rumen microbiota in dairy cattle using pyrosequencing

The objective of this study was to evaluate the changes in bacterial populations in the rumen of dairy cattle following adaptation to subacute ruminal acidosis (SARA) using 16S rRNA gene pyrosequencing. Rumen contents were collected from four cattle adapted to either a 40% (control diet, COD) or 70% (SARA induction diet, SAID) concentrate feeds. DNA was extracted from each of the samples. Bacterial 16S rRNA genes of ruminal DNA extracts were PCR amplified with 2 bar coded primer sets and sequenced by 454 pyrosequencing. At a high taxonomic level, the percentage of Proteobacteria and Bacteroidetes were reduced by SAID feeding, whereas Firmicutes and Actinobacteria were more abundant in the SAID than in the COD group. At the genus level, as compared with the COD group, the abundances of Prevotella, Treponema, Anaeroplasma, Papillibacter, Acinetobacter and unclassified populations including unclassified Lentisphaerae, and unclassified bacteria were lower (P < 0.05), while the percentages of Ruminococcus, Atopobium, unclassified Clostridiales and Bifidobacterium were increased (P < 0.05) in the SAID group. Feeding of SAID reduced (P < 0.001) the diversity of the rumen microbial community. Taken together, our findings provide a comprehensive picture of current knowledge of the community structure of the rumen bacterial ecosystem during SARA, and enhance our understanding about the ruminal microbial ecology that may be useful in the prevention of ruminal acidosis.

Changes in feed intake, nutrient digestion, plasma metabolites, and oxidative stress parameters in dairy cows with subacute ruminal acidosis and its regulation with pelleted beet pulp

The objectives of this study were to 1) determine the variation of nutrient digestion, plasma metabolites and oxidative stress parameters triggered by induced subacute ruminal acidosis (SARA); and 2) evaluate the ability of pelleted beet pulp (BP) as a replacement for ground corn to alleviate SARA. Eight Holstein-Friesian cows were fed four diets during four successive17-day periods: 1) total mixed ration (TMR) containing 0% finely ground wheat (FGW) (W0); 2) TMR containing 10% FGW (W10); 3) TMR containing 20% FGW (W20); and 4) TMR containing 10% BP as a replacement for 10% ground corn (BP10). The SARA induction protocol reduced the mean ruminal pH from 6.37 to 5.94, and the minimum ruminal pH decreased from 5.99 to 5.41 from baseline to challenge period. Mean ruminal pH increased from 5.94 to 6.05, and minimum daily ruminal pH increased from 5.41 to 5.63, when BP was substituted for corn. The apparent digestibility of nutrients was not affected by the dietary treatments, except that the digestibility of neutral detergent fibre (NDF) and acid detergent fibre (ADF) was reduced in cows fed the W20 diet compared with cows fed the W0 and W10 diets, and cows fed the BP10 diet had higher NDF and ADF digestibility than the cows fed the W20 diet. Cows fed the W20 diet had a lower plasma concentration of β-hydroxybutyrate (BHBA), non-esterified fatty acids (NEFA), cholesterol, triglyceride, and total antioxidative capacity (TAC), and a higher plasma concentration of glucose, insulin, malonaldehyde (MDA), super oxygen dehydrogenises (SOD), and glutathione peroxidase (GSH-Px) than cows fed the W0 diet. Substitution of BP for corn increased concentrations of plasma BHBA and TAC, but decreased concentrations of plasma MDA. Our results indicate that reduction of fibre digestion; the concomitant increase of plasma glucose and insulin; the decrease of plasma BHBA, NEFA, cholesterol, and triglyceride; and changes of plasma oxidative stress parameters are highly related to SARA induced by W20 diets. These variables may be alternative candidates for SARA diagnosis. We also suggest that the substitution of BP for corn could reduce the risk of SARA, increase fibre digestion, and improve the antioxidant status in dairy cows.

Grain-based versus alfalfa-based subacute ruminal acidosis induction experiments: Similarities and differences between changes in milk fatty acids

Subacute ruminal acidosis (SARA) is one of the most important metabolic disorders, traditionally characterized by low rumen pH, which might be induced by an increase in the dietary proportion of grains as well as by a reduction of structural fiber. Both approaches were used in earlier published experiments in which SARA was induced by replacing part of the ration by a grain mixture or alfalfa hay by alfalfa pellets. The main differences between both experiments were the presence of blood lipopolysaccharide and Escherichia coli and associated effects on the rumen microbial population in the rumen of grain-based induced SARA animals as well as a great amount of quickly fermentable carbohydrates in the grain-based SARA induction experiment. Both induction approaches changed rumen pH although the pH decrease was more substantial in the alfalfa-based SARA induction protocol. The goal of the current analysis was to assess whether both acidosis induction approaches provoked similar shifts in the milk fatty acid (FA) profile. Similar changes of the odd- and branched-chain FA and the C18 biohydrogenation intermediates were observed in the alfalfa-based SARA induction experiment and the grain-based SARA induction experiment, although they were more pronounced in the former. The proportion of trans-10 C18:1 in the last week of the alfalfa-based induction experiment was 6 times higher than the proportion measured during the control week. The main difference between both induction experiments under similar rumen pH changes was the decreasing sum of iso FA during the grain-based SARA induction experiment whereas the sum of iso FA remained stable during the alfalfa-based SARA induction experiment. The cellulolytic bacterial community seemed to be negatively affected by either the presence of E. coli and the associated lipopolysaccharide accumulation in the rumen or by the amount of starch and quickly fermentable carbohydrates in the diet. In general, changes in the milk FA profile were related to changes in rumen pH. Nevertheless, feed characteristics (low in structural fiber vs. high in starch) also affected the milk FA profile and, as such, both effects should be taken into account when subacute acidosis occurs.

Effects of Increasing Non-Fiber Carbohydrate to Neutral Detergent Fiber Ratio on Rumen Fermentation and Microbiota in Goats

Six rumen-cannulated lactating Guanzhong goats were used to investigate changes in ruminal fermentation pattern and the microbiota following a subacute ruminal acidosis (SARA) inducing procedure. Induction of SARA was performed by increasing dietary non-fiber carbohydrate (NFC) to neutral detergent fiber (NDF) ratio from 1.02 (stage 1) to 1.24 (stage 2), 1.63 (stage 3) and 2.58 (stage 4). A dynamic pH monitoring system, real-time fluorescent quantitative PCR and conventional anaerobic culture were used to assess changes in ruminal pH and microbiota. Results indicated that rumen fermentation patterns changed significantly with increased NFC: NDF ratio. The decline in ruminal pH was caused by increased ruminal total volatile fatty acids (TVFA), which was mainly attributed to a significant increase in ruminal butyrate, rather than the accumulation of ruminal lactic acid. In addition, in the course of SARA, the number of rumen microoganisms altered significantly, with increases in ruminal amylolytic bacteria, Lactobacilli, Streptococcus bovis and Megasphaera elsdenii, the latter particularly dramatically indicating that it may be the main factor responsible for the increase in butyrate, and decrease in protozoa.

Subacute ruminal acidosis (SARA), endotoxins and health consequences

Several symptoms of subacute ruminal acidosis (SARA) in cattle, including inflammation, have been attributed to the translocation of free bacterial lipopolysaccharide endotoxin (LPS) from the rumen into the interior circulation. The objective of this review is to assess evidence for this effect of LPS. Induction of SARA using grain-based SARA challenges increases LPS in the rumen, caecum, and feces. However, SARA induced using a nutritional challenge based on feeding pellets of ground alfalfa (alfalfa-pellet SARA challenge) increases LPS in the rumen, but not in the large intestine. This difference between the two SARA challenges may be due to the increases in starch and fermentation in the large intestine that only occur during grain-based SARA challenges.Grain-based SARA challenges have repeatedly been associated with increases in acute phase proteins in peripheral blood, which shows that these challenges cause an innate immune response. This may be the result of translocation of LPS. However, as inflammation of the epithelium of the digestive tract also causes an acute phase response, the acute phase response could have occurred without this translocation. Despite similar responses to grain-based and alfalfa-pellet SARA challenges in terms of rumen pH depression and rumen LPS concentration, only the grain-based SARA challenge causes an acute phase response. This shows that the combination of low rumen pH and high rumen LPS alone do not cause translocation and an acute phase response.Although high luminal LPS can reduce the barrier function of the rumen epithelium, this is unlikely the major cause of LPS translocation. High acidity of digesta in the large intestine may reduce the barrier function of its epithelium, and thereby cause the translocation of LPS. Despite this, evidence for increases in the concentration of LPS in peripheral blood during SARA is limited. This could be the result of inadequate sensitivities of the tests used for LPS measurement in blood, and the fast clearance rate of LPS from portal and peripheral blood.The symptoms of SARA vary depending on the method used for the SARA challenge, even if these challenges result in similar rumen pH depressions. This may be explained by effects of these challenge on digesta other than the acidity, such as the starch content of digesta in the rumen and in the large intestine.

The diversity of the fecal bacterial community and its relationship with the concentration of volatile fatty acids in the feces during subacute rumen acidosis in dairy cows

BackgroundSub-acute ruminal acidosis (SARA) is a well-recognized digestive disorder found in particular in well-managed dairy herds. SARA can result in increased flow of fermentable substrates to the hindgut, which can increase the production of volatile fatty acids, alter the structure of the microbial community, and have a negative effect on animal health and productivity. However, little is known about changes in the structure of the microbial community and its relationship with fatty acids during SARA. Four cannulated primiparous (60 to 90 day in milk) Holstein dairy cows were assigned to two diets in a 2 × 2 crossover experimental design. The diets contained (on a dry matter basis): 40% (control diet, COD) and 70% (SARA induction diet, SAID) concentrate feeds. Samples of ruminal fluid and feces were collected on day 12, 15, 17 and 21 of the treatment period, and the pH was measured in the ruminal and fecal samples; the fecal microbiota was determined by pyrosequencing analysis of the V1–V3 region of amplified 16S ribosomal RNA (16S rRNA).ResultsSAID decreased ruminal and fecal pH and increased the propionate, butyrate and total volatile fatty acid (TVFA) concentration in feces when compared with the COD. A barcoded DNA pyrosequencing method was used to generate 2116 16S operational taxonomic units (OTUs). A total of 11 phyla were observed, distributed amongst all cattle on both diets; however, only 5 phyla were observed in all animals regardless of dietary treatment, and considerable animal to animal variation was revealed. The average abundance and its range of the 5 phyla were as follows: Firmicutes (63.7%, 29.1–84.1%), Proteobacteria (18.3%, 3.4–46.9%), Actinobacteria (6.8%, 0.4–39.9%), Bacteroidetes (7.6%, 2.2–17.7%) and Tenericutes (1.6%, 0.3–3%). Feeding the SAID resulted in significant shifts in the structure of the fecal microbial community when compared with the traditional COD. Among the 2116 OTUs detected in the present study, 88 OTUs were affected significantly by diet; and the proportion of these OTUs was 20.6% and 17.4% among the total number of sequences, respectively. Among the OTUs affected, the predominant species, including OTU2140 (G: Turicibacter), OTU1695 (G: Stenotrophomonas) and OTU8143 (F: Lachnospiraceae), were increased, while the abundance of OTU1266 (S: Solibacillus silvestris) and OTU2022 (G: Lysinibacillus) was reduced in the SAID group compared with the COD. Further, our results indicated that the fecal volatile fatty acid (VFA) concentrations were significantly related to presence of some certain species of Bacteroidetes and Firmicutes in the feces.ConclusionsThis is, to our knowledge, the first study that has used barcoded DNA pyrosequencing to survey the fecal microbiome of dairy cattle during SARA. Our results suggest that particular bacteria and their metabolites in the feces appear to contribute to differences in host health between those given SAID and traditional COD feeding. A better understanding of these microbial populations will allow for improved nutrient management and increased animal growth performance.

A single mild episode of subacute ruminal acidosis does not affect ruminal barrier function in the short term

Twenty-four German Merino sheep (72.3±10.1kg of body weight) were fed an all-hay diet and assigned to either the subacute ruminal acidosis (SARA) treatment (n=17) or sham treatment (n=7). The SARA sheep were orally dosed with a 2.2 M glucose solution to supply 5g of glucose/kg of body weight, whereas sham sheep received an equal volume of water. Ruminal pH was measured for 48h before and 3h after the oral dose. Sheep were then killed and ruminal epithelia from the ventral sac were mounted in Ussing chambers. The serosal-to-mucosal flux rate of partially 3H-labeled mannitol (Jmannitol-SM), an indicator of barrier function, was measured while epithelia were exposed to 3 sequential in vitro measurement periods lasting 1h each. The measurement periods consisted of baseline, challenge, and recovery periods and were interspersed by 30-min periods for treatment equilibration. Baseline conditions were pH 6.1 (mucosal solution) and pH 7.4 (serosal solution) with a bilateral osmolarity of 293mOsm/L. During the challenge period, the mucosal side of the epithelia was exposed to either an acidotic challenge (pH 5.2, osmolarity 293mOsm/L) or an osmotic challenge (pH 6.1, osmolarity 450mOsm/L); a third group served as control (pH 6.1, osmolarity 293mOsm/L). The mucosal buffer solution was replaced for the recovery period. In vivo, sheep on the SARA treatment had lower mean (5.77 vs. 6.67) and nadir (5.48 vs. 6.47) ruminal pH for the 3h following the oral drench compared with sham sheep, indicating the successful induction of SARA with the oral glucose dose. Despite the marked reduction in pH in vivo, induction of SARA had no detectable effects on the baseline measurements of Jmannitol-SM, tissue conductance (Gt), and short-circuit current (Isc) in vitro. However, reducing mucosal pH to 5.2 in vitro had negative effects on epithelial barrier function in the recovery period, including increased Jmannitol-SM, increased Gt, and decreased Isc. The osmotic challenge increased Jmannitol-SM and Gt and decreased Isc during the challenge period, which was reversible in the recovery period except for slight reduction in Isc. Interactions between the in vitro treatment and measurement period were detected for Jmannitol-SM, Gt, and Isc. These data indicate that a mild episode of SARA (nadir pH, 5.48; duration ruminal pH <5.8, 111min relative to the 180-min measurement period) does not affect ruminal epithelial barrier function immediately after the episode but that a rapid and more severe acidification (pH 5.2) in vitro increases epithelial permeability following the insult.

Epithelial Capacity for Apical Uptake of Short Chain Fatty Acids Is a Key Determinant for Intraruminal pH and the Susceptibility to Subacute Ruminal Acidosis in Sheep ,

Subacute ruminal acidosis (SARA) is a common digestive disorder occurring in ruminants, with considerable variation in the severity of SARA observed among animals fed the same diet. Our aim in this study was to determine whether differences in the capacity of the ruminal epithelium for the apical uptake of acetate and butyrate (determined in Ussing chambers after slaughter) explains differences observed for the severity of a preceding episode of SARA in vivo. Adult sheep with an indwelling small ruminant ruminal pH measurement system (SRS) were randomly assigned to either a SARA induction treatment (oral drench containing 5 g glucose/kg body weight; n = 17) or a sham treatment (SHAM; n = 7; 12 mL water/kg body weight). Sheep receiving the glucose drench were further classified as nonresponders (NR; n = 7) or responders (RES; n = 7) according to their ruminal pH profile for the 3 h following the oral drench. Mean ruminal pH for the 3 h following the drench differed among groups (P < 0.001), with it being highest for SHAM (6.67 ± 0.08), intermediate for NR (5.97 ± 0.05), and lowest for RES (5.57 ± 0.08) sheep. The apical uptake of acetate and butyrate did not differ between SHAM and RES sheep. However, NR sheep had greater in vitro apical uptake of acetate and butyrate and a higher plasma β-hydroxybutyrate concentration than RES sheep, suggesting greater absorptive capacity for NR. Differences between NR and RES were attributed to greater bicarbonate-independent, nitrate-sensitive uptake of acetate (P = 0.007), a tendency for greater bicarbonate-dependent uptake of acetate (P = 0.071), and greater bicarbonate-independent uptake of butyrate (P = 0.022). These data indicate that differences in the rates and pathways for the uptake of acetate and butyrate explain a large proportion of the individual variation observed for the severity of SARA.

Evaluation of the protective effect of probiotics fed to dairy cows during a subacute ruminal acidosis challenge

Subacute ruminal acidosis (SARA) can be extremely costly when it occurs in dairy cows. The use of probiotic supplements to stabilize rumen pH during the transition period following parturition could attenuate the symptoms of this metabolic disorder. Four ruminally fistulated Holstein dairy cows in mid to late lactation were assigned to the following experimental treatments in a 4 × 4 Latin square design: (1) control (C); (2) 0.6 g/cow/d of a fermentation extract of Aspergillus oryzae (AO-0.6); (3) 3 g/cow/d of A. oryzae (AO-3.0); and (4) 2 g/cow/d of a probiotic combination consisting of Enterococcus faecium and Saccharomyces cerevisiae (ES). Each period of the Latin square consisted of three weeks of adaptation to the respective treatments followed by four days of induction of SARA and three days of rest. During the four days of induction of SARA, 0.30 of ad libitum intake of the total mixed ration (TMR) was replaced with wheat and barley pellets (WBP) containing 0.5 ground wheat and 0.5 ground barley. Ruminal pH was recorded continuously using an indwelling pH probe over a 24 h period for each week of the adaptation period and continuously over the four days of SARA induction. Average ruminal pH was lower ( P=0.0001) during SARA than during the weeks of adaptation (5.6 versus 6.1). When the cows were fed ES, the minimum pH recorded was higher ( P=0.05) (5.0) than when they were in the control (4.4). The proportion of time during which pH was between 5.6 and 6.0 was higher ( P=0.05) with ES (0.31) than with C (0.22). Mean pH tended ( P=0.06) to be higher with ES (5.84) than with C (5.41), and the proportion of time during which pH was below 5.2 tended ( P=0.09) to be lower with ES (0.09) than with C (0.31). AO-0.6 tended ( P=0.07) to increase minimum pH (4.96) compared with C (4.43) and tended ( P=0.08) to decrease the percentage of time during which pH was below 5.2 (0.08 versus 0.31 for AO-0.6 and C, respectively). The addition of probiotics during SARA had no effect on the number of cellulolytic bacteria or on the population of Megasphaera elsdenii in the rumen.