Induction of Subacute Ruminal Acidosis Affects the Ruminal Microbiome and Epithelium.
ABSTRACT: 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.
Project description:Subacute ruminal acidosis (SARA) is a metabolic disease in high-producing dairy cattle, and is accompanied by rumenitis. However, the mechanism of rumenitis remains unclear. Therefore, the aim of this study was to investigate the molecular mechanism of rumenitis in dairy cows with SARA.The results showed that SARA cows displayed high concentrations of ruminal volatile fatty acids, lactic acid and lipopolysaccharide (LPS). Furthermore, the blood concentrations of LPS and acute phase proteins haptoglobin, serum amyloid-A, and LPS binding protein were significantly higher in SARA cows than in control cows. Importantly, the phosphorylation levels of nuclear factor-kappaB (NF-?B) p65, inhibitor of NF-?B (I?B), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2) were significantly higher in the rumen epithelium of SARA cows than those of control cows. The ruminal mRNA and protein levels of NF-?B- and mitogen-activated protein kinase (MAPK)s -regulated inflammatory cytokines, tumor necrosis factor ? (TNF-?), interleukin 6 (IL-6) and interleukin 1? (IL-1?), were markedly higher in SARA cows than in control cows. Similarly, serum concentrations of TNF-? and IL-6 were also significantly higher in SARA cows.These results indicate that SARA results in high concentration of ruminal LPS, which over activates the NF-?B and MAPKs inflammatory pathways and then significantly increases the expression and synthesis of pro-inflammation cytokines in the rumen epithelium, thereby partly inducing rumenitis.
Project description:Subacute ruminal acidosis (SARA) represents one of the most important digestive disorders in intensive dairy farms, and dairy cows are individually different in the severity of SARA risk. The objectives of the current study were to investigate differences in the ruminal bacterial community and metabolome in dairy cattle with different susceptibility to SARA. In the present study, 12 cows were initially enrolled in the experiment. Based on average ruminal pH, 4 cows with the lowest ruminal pH were assigned to the susceptible group (SUS, pH = 5.76, <i>n</i> = 4) and 4 cows with the highest ruminal pH assigned to the tolerant group (TOL, pH = 6.10, <i>n</i> = 4). Rumen contents from susceptible (SUS, <i>n</i> = 4) and tolerant (TOL, <i>n</i> = 4) dairy cows were collected through rumen fistula to systematically reveal the rumen microbial and metabolic alterations of dairy cows with different susceptibility to SARA using multi-omics approaches (16S and 18S rRNA gene sequencing and metabolome). The results showed that despite being fed the same diet, SUS cows had lower ruminal pH and higher concentrations of total volatile fatty acids (VFA) and propionate than TOL cows (<i>P</i> < 0.05). No significant differences were observed in dry matter intake, milk yield, and other milk compositions between the SUS and TOL groups (<i>P</i> > 0.05). The principal coordinates analysis based on the analysis of molecular variance indicated a significant difference in bacterial composition between the two groups (<i>P</i> = 0.01). More specifically, the relative abundance of starch-degrading bacteria (<i>Prevotella</i> spp.) was greater (<i>P</i> < 0.05), while the proportion of fiber-degrading bacteria (unclassified Ruminococcaceae spp., <i>Ruminococcus</i> spp., <i>Papillibacter</i>, and unclassified Family_XIII) was lower in the rumen of SUS cows compared with TOL cows (<i>P</i> < 0.05). Community analysis of protozoa showed that there were no significant differences in the diversity, richness, and community structure (<i>P</i> > 0.05). Metabolomics analysis revealed that the concentrations of organic acids (such as lactic acid), biogenic amines (such as histamine), and bacterial degradation products (such as hypoxanthine) were significantly higher in the SUS group compared to the TOL group (<i>P</i> < 0.05). These findings revealed that the higher proportion of starch-degrading bacteria/lower fiber-degrading bacteria in the rumen of SUS cows resulted in higher VFA-producing capacity, in particular propionate. This caused a disruption in metabolic homeostasis in the rumen which might be the reason for the higher susceptibility to SARA. Overall, these findings enhanced our understanding of the ruminal microbiome and metabolic changes in cows susceptible to SARA.
Project description:We investigated changes in rumen fermentation, peripheral blood metabolites and hormones, and hepatic transcriptomic dynamics in Holstein cows with and those without subacute ruminal acidosis (SARA) during the periparturient period. Overall design: SARA was diagnosed when ruminal pH was < 5.6 for more than 3 h per day (Gozho et al., 2005) during the 2 weeks following parturition. Based on these criteria, cows were assigned to the SARA (n = 8) or Control (n = 8) group.
Project description:BACKGROUND:Overfeeding of high-concentrate diet (HC) frequently leads to subacute ruminal acidosis (SARA) in modern dairy cows' production. Thiamine supplementation has been confirmed to attenuate HC induced SARA by increasing ruminal pH and ratio of acetate to propionate, and decreasing rumen lactate, biogenic amines and lipopolysaccharide (LPS). The effects of thiamine supplementation in HC on rumen bacteria and fungi profile had been detected in our previous studies, however, effects of thiamine supplementation in HC on rumen non-methanogen archaea is still unclear. The objective of the present study was therefore to investigate the effects of thiamine supplementation on ruminal archaea, especially non-methanogens in HC induced SARA cows. RESULTS:HC feeding significantly decreased dry matter intake, milk production, milk fat content, ruminal pH and the concentrations of thiamine and acetate in rumen fluid compared with control diet (CON) (P?<?0.05), while the concentrations of propionate and ammonia-nitrogen (NH3-N) were significantly increased compared with CON (P?<?0.05). These changes caused by HC were inversed by thiamine supplementation (P?<?0.05). The taxonomy results showed that ruminal archaea ranged from 0.37 to 0.47% of the whole microbiota. Four characterized phyla, a number of Candidatus archaea and almost 660 species were identified in the present study. In which Euryarchaeota occupied the largest proportion of the whole archaea. Furthermore, thiamine supplementation treatment significantly increased the relative abundance of non-methanogens compared with CON and HC treatments. Thaumarchaeota was increased in HC compared with CON. Thiamine supplementation significantly increased Crenarchaeota, Nanoarchaeota and the Candidatus phyla, however decreased Thaumarchaeota compared with HC treatment. CONCLUSIONS:HC feeding significantly decreased ruminal pH and increased the content of NH3-N which led to N loss and the increase of the relative abundance of Thaumarchaeota. Thiamine supplementation increased ruminal pH, improved the activity of ammonia utilizing bacteria, and decreased Thaumarchaeota abundance to reduce the ruminal NH3 content and finally reduced N loss. Overall, these findings contributed to the understanding of thiamine's function in dairy cows and provided new strategies to improve dairy cows' health under high-concentrate feeding regime.
Project description:Introduction:Thiamine is known to attenuate high-concentrate diet induced subacute ruminal acidosis (SARA) in dairy cows, however, the underlying mechanisms remain unclear. Objectives:The major objective of this study was to investigate the metabolic mechanisms of thiamine supplementation on high-concentrate diet induced SARA. Methods:Six multiparous, rumen-fistulated Holstein cows were used in a replicated 3?×?3 Latin square design. The treatments included a control diet (CON; 20% starch, dry matter basis), a SARA-inducing diet (SAID; 33.2% starch, dry matter basis) and SARA-inducing diet supplemented with 180 mg of thiamine/kg of dry matter intake (SAID?+?T). On d21 of each period, ruminal fluid samples were collected at 3 h post feeding, and GC/MS was used to analyze rumen fluid samples. Results:PCA and OPLS-DA analysis demonstrated that the ruminal metabolite profile were different in three treatments. Compared with CON treatment, SAID feeding significantly decreased rumen pH, acetate, succinic acid, increased propionate, pyruvate, lactate, glycine and biogenic amines including spermidine and putrescine. Thiamine supplementation significantly decreased rumen content of propionate, pyruvate, lactate, glycine and spermidine; increase rumen pH, acetate and some medium-chain fatty acids. The enrichment analysis of different metabolites indicated that thiamine supplementation mainly affected carbohydrates, amino acids, pyruvate and thiamine metabolism compared with SAID treatment. Conclusions:These findings revealed that thiamine supplementation could attenuate high-concentrate diet induced SARA by increasing pyruvate formate-lyase activity to promote pyruvate to generate acetyl-CoA and inhibit lactate generation. Besides, thiamine reduced biogenic amines to alleviate ruminal epithelial inflammatory response.
Project description:Background:Residual feed intake (RFI) in dairy cattle typically calculated at peak lactation is a measure of feed efficiency independent of milk production level. The objective of this study was to evaluate differences in ruminal bacteria, biopolymer hydrolyzing enzyme activities, and overall performance between the most- and the least-efficient dairy cows during the peripartal period. Twenty multiparous Holstein dairy cows with daily ad libitum access to a total mixed ration from d - 10 to d 60 relative to the calving date were used. Cows were classified into most-efficient (i.e. with low RFI, n = 10) and least-efficient (i.e. with high RFI, n = 10) based on a linear regression model involving dry matter intake (DMI), fat-corrected milk (FCM), changes in body weight (BW), and metabolic BW. Results:The most-efficient cows had ~ 2.6 kg/d lower DMI at wk 4, 6, 7, and 8 compared with the least-efficient cows. In addition, the most-efficient cows had greater relative abundance of total ruminal bacterial community during the peripartal period. Compared with the least-efficient cows, the most-efficient cows had 4-fold greater relative abundance of Succinivibrio dextrinosolvens at d - 10 and d 10 around parturition and tended to have greater abundance of Fibrobacter succinogenes and Megaspheara elsdenii. In contrast, the relative abundance of Butyrivibrio proteoclasticus and Streptococcus bovis was lower and Succinimonas amylolytica and Prevotella bryantii tended to be lower in the most-efficient cows around calving. During the peripartal period, the most-efficient cows had lower enzymatic activities of cellulase, amylase, and protease compared with the least-efficient cows. Conclusions:The results suggest that shifts in ruminal bacteria and digestive enzyme activities during the peripartal period could, at least in part, be part of the mechanism associated with better feed efficiency in dairy cows.
Project description:Ruminal thiamine deficiencies occur when dairy cows are overfed with high-concentrate diet, and thiamine supplementation has been proved to attenuate high-concentrate diet induced SARA. However, there is limited knowledge of the relationship between thiamine supplementation in high-concentrate diets and ruminal fungi. In order to investigate the impacts of thiamine supplementation on ruminal fungi, twelve Chinese Holstein dairy cows were randomly assigned into three treatments: control diet (CON; 20% starch, dry matter basis), high-concentrate diet (HC; 33.2% starch, dry matter basis) and high-concentrate diet supplemented with 180 mg thiamine/kg dry matter intake. Dry matter intake and milk production were recorded during the experimental periods. On day 21, rumen fluid samples were collected at 3 h postfeeding and ruminal pH, thiamine concentration and volatile fatty acids were measured. Metagenome sequencing method was conducted to detect ruminal fungi composition. Feeding HC significantly decreased dry matter intake, milk production, ruminal pH, ruminal acetate and thiamine concentration, however, significantly increased propionate and isovalerate (P?<?0.05). These changes were inversed by thiamine supplementation (P?<?0.05). Totally, seven phyla and almost 1050 species of rumen fungi were identified across all samples in which especially, 3 genera and 10 species of strictly anaerobic fungi phylum Neocallimastigomycota was found. Principal coordinate analysis indicated that feeding HC and thiamine supplementation caused a significant inverse in ruminal fungi composition. Feeding HC significantly decreased the abundance of fungi compared with CON (P?<?0.05) while thiamine supplementation significantly increased the abundance of ruminal fungi (P?<?0.05). These results indicated that thiamine supplementation may effectively attenuate rumen metabolic disorder caused by HC diet through buffering the ruminal pH, shifting the rumen fermentation pattern and increasing the abundance of ruminal fungi. The findings in this study could therefore contribute to the further understanding of the mechanism of thiamine's function in dairy cows.
Project description: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.
Project description:The impact of a long-term subacute rumen acidosis (SARA) on the bovine epimural bacterial microbiome (BEBM) and its consequences for rumen health is poorly understood. This study aimed to investigate shifts in the BEBM during a long-term transient SARA model consisting of two concentrate-diet-induced SARA challenges separated by a 1-week challenge break. Eight cows were fed forage and varying concentrate amounts throughout the experiment. In total, 32 rumen papilla biopsies were taken for DNA isolation (4 sampling time points per cow: at the baseline before concentrate was fed, after the first SARA challenge, after the challenge break, and after the second SARA challenge). Ruminal pH was continuously monitored. The microbiome was determined using Illumina MiSeq sequencing of the 16S rRNA gene (V345 region). In total 1,215,618 sequences were obtained and clustered into 6833 operational taxonomic units (OTUs). Campylobacter and Kingella were the most abundant OTUs (16.5 and 7.1%). According to ruminal pH dynamics, the second challenge was more severe than the first challenge. Species diversity estimates and evenness increased during the challenge break compared to all other sampling time points (P < 0.05). During both SARA challenges, Kingella- and Azoarcus-OTUs decreased (0.5 and 0.4 fold-change) and a dominant Ruminobacter-OTU increased during the challenge break (18.9 fold-change; P < 0.05). qPCR confirmed SARA-related shifts. During the challenge break noticeably more OTUs increased compared to other sampling time points. Our results show that the BEBM re-establishes the baseline conditions slower after a SARA challenge than ruminal pH. Key phylotypes that were reduced during both challenges may help to establish a bacterial fingerprint to facilitate understanding effects of SARA conditions on the BEBM and their consequences for the ruminant host.
Project description:Milk fat depression (MFD) syndrome represents a significant drawback to the dairy industry. The aim of this study was to unravel the ruminal metabolome-microbiome interaction in response to diet-induced MFD in dairy cows. Twelve healthy second parity Holstein dairy cows (days in milk (DIM) = 119 ± 14) were randomly assigned into control (CON, n = 6) group and treatment (TR, n = 6) group. Cows in TR group received a high-starch total mixed ration (TMR) designed to induce an MFD syndrome. Decreased milk fat yield and concentration in TR cows displayed the successful development of MFD syndrome. TR diet increased the relative abundance of Prevotella and decreased the relative abundance of unclassified Lachnospiraceae, Oribacterium, unclassified Veillonellaceae and Pseudobutyrivibrio in ruminal fluid. Metabolomics analysis revealed that the ruminal fluid content of glucose, amino acids and amines were significantly increased in TR cows compared with CON cows. Correlation analysis revealed that the concentration of amines and amino acids were highly correlated with the abundance of Oribacterium, Pseudobutyrivibrio, RC9_gut_group, unclassified BS11_gut_group and Selenomonas. In general, these findings revealed that TR diet reduced the rumination time and altered rumen fermentation type, which led to changes in the composition of ruminal microbiota and metabolites, and caused MFD.