Epigallocatechin-3-gallate protects bovine ruminal epithelial cells against lipopolysaccharide-induced inflammatory damage by activating autophagy.

BACKGROUND: Subacute ruminal acidosis (SARA) causes an increase in endotoxin, which can induce immune and inflammatory responses in the ruminal epithelium of dairy cows. In non-ruminants, epigallocatechin-3-gallate (EGCG), a major bioactive ingredient of green tea, is well-known to alleviate inflammation. Whether EGCG confers protection against SARA-induced inflammation and the underlying mechanisms are unknown. RESULTS: In vivo, eight ruminally cannulated Holstein cows in mid-lactation were randomly assigned to either a low-concentrate (40%) diet (CON) or a high-concentrate (60%) diet (HC) for 3 weeks to induce SARA (n = 4). Cows with SARA had greater serum concentrations of tumor necrosis factor (TNF)-α and interleukin-6, and epithelium had histological signs of damage. In vitro, immortalized bovine ruminal epithelial cells (BREC) were treated with lipopolysaccharide (LPS) to imitate the inflammatory damage caused by SARA. Our data revealed that BREC treated with 10 µg/mL LPS for 6 h successfully induce a robust inflammatory response as indicated by increased phosphorylation of IκBα and nuclear factor kappa-B (NF-κB) p65. Pre-treatment of BREC with 50 µmol/L EGCG for 6 h before LPS challenge promoted the degradation of NLR family pyrin domain containing 3 (NLRP3) inflammasome through activation of autophagy, which further repressed activation of NF-κB pathway targeting Toll-like receptor 4 (TLR4). Analyses also revealed that the ECGG upregulated tight junction (TJ) protein expression upon incubation with LPS. CONCLUSIONS: Subacute ruminal acidosis causes ruminal epithelium injury and systemic inflammation in dairy cows. However, the anti-inflammatory effects of EGCG help preserve the integrity of the epithelial barrier through activating autophagy when BREC are exposed to LPS. Thus, EGCG could potentially serve as an effective therapeutic agent for SARA-associated inflammation.

Predicting subacute ruminal acidosis from milk mid-infrared estimated fatty acids and machine learning on Canadian commercial dairy herds.

Our objective was to validate the possibility of detecting SARA from milk Fourier transform mid-infrared spectroscopy estimated fatty acids (FA) and machine learning. Subacute ruminal acidosis is a common condition in modern commercial dairy herds for which the diagnostic remains challenging due to its symptoms often being subtle, nonexclusive, and not immediately apparent. This observational study aimed at evaluating the possibility of predicting SARA by developing machine learning models to be applied to farm data and to provide an estimated portrait of SARA prevalence in commercial dairy herds. A first data set composed of 488 milk samples of 67 cows (initial DIM = 8.5 ± 6.18; mean ± SD) from 7 commercial dairy farms and their corresponding SARA classification (SARA+ if rumen pH <6.0 for 300 min, else SARA-) was used for the development of machine learning models. Three sets of predictive variables: i) milk major components (MMC), ii) milk FA (MFA), and iii) MMC combined with MFA (MMCFA) were submitted to 3 different algorithms, namely Elastic net (EN), Extreme gradient boosting (XGB), and Partial least squares (PLS), and evaluated using 3 different scenarios of cross-validation. Accuracy, sensitivity, and specificity of the resulting 27 models were analyzed using a linear mixed model. Model performance was not significantly affected by the choice of algorithm. Model performance was improved by including fatty acids estimations (MFA and MMCFA as opposed to MMC alone). Based on these results, one model was selected (algorithm: EN; predictive variables: MMCFA; 60.4, 65.4, and 55.3% of accuracy, sensitivity, and specificity, respectively) and applied to a large data set comprising the first test-day record (milk major components and FA within the first 70 DIM of 211,972 Holstein cows (219,503 samples) collected from 3001 commercial dairy herds. Based on this analysis, the within-herd SARA prevalence of commercial farms was estimated at 6.6 ± 5.29% ranging from 0 to 38.3%. A subsequent linear mixed model was built to investigate the herd-level factors associated to higher within-herd SARA prevalence. Milking system, proportion of primiparous cows, herd size and seasons were all herd-level factors affecting SARA prevalence. Furthermore, milk production was positively, and milk fat yield negatively associated with SARA prevalence. Due to their moderate levels of accuracy, the SARA prediction models developed in our study, using data from continuous pH measurements on commercial farms, are not suitable for diagnostic purpose. However, these models can provide valuable information at the herd level.

Effects of pellet starch levels in automatic milking systems on rumen fermentation, plasma metabolites, and milk production in mid-lactation cows.

The aim of this study was to evaluate whether the starch levels in pellets fed to cows in automatic milking systems (AMS) affect subacute ruminal acidosis (SARA) occurrence and metabolite parameters. Twenty-four lactating cows (124.4 ± 49.9 days in milk) were studied in a crossover design with two periods of 21 days each and two treatment groups-a control group fed AMS pellets containing 30.0% of starch dry matter (DM) and an experimental group fed AMS pellets containing 23.5% of starch DM. All cows received the same partial mixed ration (PMR). The 1-hr mean ruminal pH in both groups decreased over 4 hr after feeding on PMR but recovered by the next morning. The ruminal pH was unaffected by either treatment, and both groups developed SARA. The groups had no significant differences in the concentrations of ruminal volatile fatty acids, lipopolysaccharides, plasma acute-phase proteins, other metabolites, and hormones. The milk yield and composition were not different in both groups. Feeding low-starch pellets in the AMS did not contribute to the risk of SARA occurrence in cows and had no additive effects on rumen fermentation, plasma metabolites, or milk production.

Effect of Calcareous Marine Algae Buffer on High-Producing Dairy Cows during Peak Lactation.

The aim of the study was to investigate the effect of calcareous marine algae (Lithotamium calcareum)-based rumen content buffer (CMA) included in concentrated feed within total mixed ration (TMR), fed to 34 peak lactation (87-144 days in milk) Holstein dairy cows, randomized into two groups (group A, n = 17; group B, n = 17), wearing collars with accelerometers, and housed a in barn with automatic feed-weigh troughs. During the first phase P1, group A received TMR with CMA (TMR-E) and group B was fed TMR without the buffer (TMR-C). For P2, the treatments in the groups were exchanged. Feed intake, feeding time (FT), rumination time (RT), milk yield, milk composition, and rumen pH were measured by barn technologies, and rumen fluid and feces composition were analyzed in the laboratory. Differences between the TMR-E and TMR-C in most parameters under study were statistically insignificant, except overall FT and RT, which differed significantly between the groups. Group A, feeding at P1 by TMR-E, exhibited higher FT and RT than Group B (202 min/cow/day vs. 184 min/cow/day, and 486 min/cow/day vs. 428 min/cow/day, respectively). The RT significantly increased after switching from TMR-C to TMR-E. This implies that the buffer effect is delayed and persists after the withdrawal. In the group of cows that received control TMR without buffer in the first phase, RT and milk protein content increased significantly in the first week after the addition of buffer.

Development of an experimental model for liver abscess induction in Holstein steers using an acidotic diet challenge and bacterial inoculation.

Holstein steers (n = 40; initial BW = 84.9 ±â€…7.1 kg) were used to study the genesis of liver abscesses (LA) using an acidotic diet challenge with or without intraruminal bacterial inoculation. Steers were housed in individual pens inside a barn and randomly assigned to one of three treatments: (1) low-starch control diet comprised primarily of dry-rolled corn and wet corn gluten feed (CON); (2) high-starch acidotic diet with steam-flaked corn (AD); or (3) acidotic diet plus intraruminal inoculation with Fusobacterium necrophorum subsp. necrophorum (9.8 × 108 colony forming units [CFU]/mL), Trueperella pyogenes (3.91 × 109 CFU/mL), and Salmonella enterica serovar Lubbock (3.07 × 108 CFU/mL), previously isolated from LA (ADB). Steers in AD and ADB were fed the acidotic diet for 3 d followed by 2 d of the CON diet, and this cycle was repeated four times. On day 23, ADB steers were intraruminally inoculated with the bacteria. At necropsy, gross pathology of livers, lungs, rumens, and colons was noted. Continuous data were analyzed via mixed models as repeated measures over time with individual steer as the experimental unit. Mixed models were also used to determine the difference in prevalence of necropsy scores among treatments. Ruminal pH decreased in AD and ADB steers during each acidotic diet cycle (P ≤ 0.05). LA prevalence was 42.9% (6 of 14) in ADB vs. 0% in AD or CON treatments (P < 0.01). Ruminal damage was 51.1% greater in ADB than in AD (P ≤ 0.04). Culture of LA determined that 100% of the abscesses contained F. necrophorum subsp. necrophorum, 0% contained T. pyogenes, 50% contained Salmonella, and 50% contained a combination of F. necrophorum subsp. necrophorum and Salmonella. The F. necrophorum subsp. necrophorum was clonally identical to the strain used for the bacterial inoculation based on phylogenetic analysis of the whole genome. This experimental model successfully induced rumenitis and LA in Holstein steers and confirms the central dogma of LA pathogenesis that acidosis and rumenitis lead to the entry of F. necrophorum into the liver to cause abscesses. Our findings suggest that an acidotic diet, in conjunction with intraruminal bacterial inoculation, is a viable model to induce LA. Further research is needed to determine the repeatability of this model, and a major application of the model will be in evaluations of novel interventions to prevent LA.

Liver abscesses (LA) in feedlots are costly to the beef industry. At harvest, LA cause an increase in liver condemnations, carcass trimming, and a decrease in quality grade. The objective of this research was to develop an experimental LA model in Holstein steers using an acidotic diet with and without intraruminal inoculation of bacteria involved in LA formation. These data suggest acidotic diet challenges in conjunction with bacterial inoculation were able to induce LA in Holstein steers. The acidotic diet alone caused reduced rumen content pH and caused rumen wall inflammation and damage, observed at harvest. Nonetheless, the addition of bacteria had a compounding effect on rumen damage. Both bacteria inoculated were isolated from 57% of LA suggesting they may work in synergy to form LA.

Subacute ruminal acidosis induces pyroptosis via the mitophagy-mediated NLRP3 inflammasome activation in the livers of dairy cows fed a high-grain diet.

High-grain (HG) feeding can trigger subacute ruminal acidosis (SARA) and subsequent liver tissue injury. This study investigated pyroptosis and NLRP3 inflammasome activation in SARA-induced liver injury, and the role of mitophagy during this process. Twelve mid-lactating Holstein cows equipped with rumen fistulas were randomly divided into 2 groups: a low-grain (LG) diet group (grain:forage = 4:6) and a HG diet group (grain:forage = 6:4). Each group had 6 cows. The experiment lasted for 3 wk. The ruminal fluid was collected through the rumen fistula on experimental d 20 and 21, and the pH immediately measured. At the end of the experiment, all animals were slaughtered, and peripheral blood and liver tissue were collected. The ruminal pH was lower in the HG group than that in the LG group at all time points. In addition, the ruminal pH in the HG group was lower than 5.6 at 3 consecutive time points after feeding (4, 6, and 8 h on d 20; 2, 4, and 6 h on d 21), indicating that HG feeding induced SARA. The content of lipopolysaccharide, IL-1ß, and apoptosis-related cysteine protease 1 (caspase-1) and the activity of alanine aminotransferase and aspartate aminotransferase in the blood plasma of the HG group were all significantly increased. Hepatic caspase-1 activity was increased in the livers of the HG group. The increased expression levels of pyroptosis- and NLRP3 inflammasome-related genes IL1B, IL18, gasdermin D (GSDMD), apoptosis-associated speck-like protein containing a card (ASC), NLR family pyrin domain-containing 3 (NLRP3), and caspase-1 (CASP1) in liver tissue of the HG group were detected. Furthermore, western blot analysis showed that HG feeding led to increased expression of pyroptosis- and NLRP3 inflammasome-related proteins GSDMD N-terminal (GSDMD-NT), IL-1ß, IL-18, cleaved-caspase-1, ASC, NLRP3, and cleaved-caspase-11 and upregulated expression of mitophagy-related proteins microtubule-associated protein 1 light chain 3 II (MAP1LC3-II), beclin 1 (BECN1), Parkin, and PTEN-induced kinase 1 (PINK1) in liver tissue. Collectively, our results revealed that SARA caused increased mitophagy and activated the NLRP3 inflammasome, causing pyroptosis and subsequent liver injury in dairy cows fed a HG diet.

Effects of ruminal lipopolysaccharide exposure on primary bovine ruminal epithelial cells.

The objective of this study was to investigate the immunopotential of ruminal lipopolysaccharides (LPS) on cultured primary bovine rumen epithelial cells (REC). Primary bovine REC were isolated from 6 yearling steers and grown in culture for 3 experiments. Experiment 1 aimed to determine the immunopotential of ruminal LPS, experiment 2 aimed to assess tolerance to chronic LPS exposure, and experiment 3 aimed to evaluate antagonistic interactions between ruminal and Escherichia coli LPS. In experiments 1 and 2, REC were exposed to nonpyrogenic water, 20 µg/mL E. coli LPS (EC20), 10 µg/mL ruminal LPS, 20 µg/mL ruminal LPS, and 40 µg/mL ruminal LPS, either continuously or intermittently. For the continuous exposure, REC underwent a 6 h exposure, whereas for the intermittent exposure, the procedure was: (1) a 12 h continuous exposure to treatments followed by LPS removal for 24 h and then another 12 h of exposure (RPT), and (2) a 12 h continuous exposure to treatments followed by LPS removal and a recovery period of 36 h (RCV). In experiment 3, REC were exposed to nonpyrogenic water, 1 µg/mL E. coli LPS, 1 µg/mL ruminal LPS to 1 µg/mL E. coli LPS, 10 µg/mL ruminal LPS to 1 µg/mL E. coli LPS, and 50 µg/mL ruminal LPS to 1 µg/mL E. coli LPS. Each experiment was done as a complete randomized block design with 6 REC donors. The REC-donor was used as blocking factor. Each treatment had 2 technical replicates, and treatment responses for all data were analyzed with the MIXED procedure of SAS. For all experiments, total RNA was extracted from REC and real-time quantitative PCR was performed to determine the relative expression of genes for toll-like receptors (TLR2 and TLR4), proinflammatory cytokines (TNF, IL1B, and IL6), chemokines (CXCL2 and CXCL8), growth factor-like cytokines (CSF2 and TGFB1), and a lipid mediator (PTGS2). In experiment 1, the targeted genes were upregulated by EC20, whereas all ruminal LPS treatments resulted in a lower transcript abundance. Regarding RPT, and RCV condition, in experiment 2, the expression of targeted genes was not affected or was at a lower abundance to EC20 when compared with ruminal LPS treatments. Lastly, in experiment 3, all targeted genes resulted in lower or similar transcript abundance on all ruminal LPS ratios. Overall, our results indicate that ruminal LPS have a limited capacity to activate the TLR4/NF-kB pathway and to induce the expression of inflammatory genes.

Can computerized rumen mucosal colorimetry serve as an effective field test for managing subacute ruminal acidosis in feedlot cattle?

Subacute ruminal acidosis (SARA) is one of the major nutritional disorders in the dairy and beef industries, leading to significant financial losses. Diagnosing SARA is challenging due to the need to evaluate multiple parameters, such as milk fat/protein ratio, ruminal lactate, and hemogram, instead of relying on a single definitive symptom or diagnostic method. This study aimed to evaluate the effectiveness of computerized rumen colorimetry in detecting SARA in beef cattle. Over one year, 75 cattle aged 8-10 months from five farms were periodically monitored for rumen pH prior to slaughter. Samples of rumen wall and rumen content were obtained at slaughter for analysis. The study found a positive correlation coefficient between rumen pH and color components, particularly for Red (0.853) and color lightness (L) (0.862). The darkening of the rumen epithelium's color was attributed to the effect of rumen pH on the keratinized layer of the epithelium. Furthermore, an increase in the thickness of ruminal epithelium layers, particularly non-keratinized and total epithelium, was observed in animals with a history of SARA. It is possible that the lower rumen pH increases the rate of replacement of the keratinized epithelium, and the non-keratinized epithelium overgrows to compensate for the need to of produce keratinized layers. In conclusion, computerized rumen colorimetry shows promise as a reliable method for managing SARA in bovine farms by monitoring the condition in the slaughterhouse. Further research is needed to evaluate its effectiveness in detecting SARA in live animals.

Effects of feed additives on rumen function and bacterial and archaeal communities during a starch and fructose challenge.

The objective of this study was to improve understandings of the rumen microbial ecosystem during ruminal acidosis and responses to feed additives to improve prudent use strategies for ruminal acidosis control. Rumen bacterial and archaeal community composition (BCC) and its associations with rumen fermentation measures were examined in Holstein heifers fed feed additives and challenged with starch and fructose. Heifers (n = 40) were randomly allocated to 5 treatment groups: (1) control (no additives); (2) virginiamycin (VM; 200 mg/d); (3) monensin (MT; 200 mg/d) + tylosin (110 mg/d); (4) monensin (MLY; 220 mg/d) + live yeast (5.0 × 1012 cfu/d); (5) sodium bicarbonate (BUF; 200 g/d) + magnesium oxide (30 g/d). Heifers were fed twice daily a 62% forage:38% concentrate total mixed ration at 1.25% of body weight (BW) dry matter (DM)/d for a 20-d adaptation period with their additive(s). Fructose (0.1% of BW/d) was added to the ration for the last 10 d of adaptation. On d 21 heifers were challenged once with a ration consisting of 1.0% of BW DM wheat and 0.2% of BW fructose plus their additive(s). A rumen sample was collected from each heifer via stomach tube weekly (d 0, 7, 14) and 5 times over a 3.6 h period at 5, 65, 115, 165, and 215 min after consumption of the challenge ration (d 21) and analyzed for pH, and ammonia, d- and l-lactate, volatile fatty acids (VFA), and histamine concentrations and total bacteria and archaea. The 16S rRNA gene spanning the V4 region was PCR amplified and sequenced. Alpha and ß diversity and associations of relative abundances of taxa with rumen fermentation measures were evaluated. Rumen BCC shifted among treatment groups in the adaptation period and across the challenge sampling period, indicating the feed additives had different modes of action. The monensin-containing treatment groups, MT and MLY often had similar relative abundances of rumen bacterial phyla and families. The MLY treatment group was characterized in the challenge period by increased relative abundances of the lactate utilizing genera Anaerovibrio and Megasphaera. The MLY treatment group also had increased diversity of ruminal bacteria which may provide resilience to changes in substrates. The control and BUF treatment groups were most similar in BCC. A redundancy analysis showed the MLY treatment group differed from all other treatment groups and concentrations of histamine and valerate in the rumen were associated with the most variation in the microbiota, 5.3% and 4.8%, respectively. It was evident from the taxa common to all treatment groups that cattle have a core microbiota. Functional redundancy of rumen bacteria which was reflected in the greater sensitivity for the rumen BCC than rumen fermentation measures likely provide resilience to changes in substrate. This functional redundancy of microbes in cattle suggests that there is no single optimal ruminal microbial population and no universally superior feed additive(s). In summary, differences in modes of action suggest the potential for more targeted and improved prudent use of feed additives with no single feed additive(s) providing an optimal BCC in all heifers.

Sodium Propionate Relieves LPS-Induced Inflammation by Suppressing the NF-ĸB and MAPK Signaling Pathways in Rumen Epithelial Cells of Holstein Cows.

Subacute ruminal acidosis (SARA) is a prevalent disease in intensive dairy farming, and the rumen environment of diseased cows acidifies, leading to the rupture of gram-negative bacteria to release lipopolysaccharide (LPS). LPS can cause rumentitis and other complications, such as liver abscess, mastitis and laminitis. Propionate, commonly used in the dairy industry as a feed additive, has anti-inflammatory effects, but its mechanism is unclear. This study aims to investigate whether sodium propionate (SP) reduces LPS-induced inflammation in rumen epithelial cells (RECs) and the underlying mechanism. RECs were stimulated with different time (0, 1, 3, 6, 9, 18 h) and different concentrations of LPS (0, 1, 5, 10 µg/mL) to establish an inflammation model. Then, RECs were treated with SP (15, 25, 35 mM) or 10 µM PDTC in advance and stimulated by LPS for the assessment. The results showed that LPS (6h and 10 µg/mL) could stimulate the phosphorylation of NF-κB p65, IκB, JNK, ERK and p38 MAPK through TLR4, and increase the release of TNF-α, IL-1ß and IL-6. SP (35 mM) can reduce the expression of cytokines by effectively inhibiting the NF-κB and MAPK inflammatory pathways. This study confirmed that SP inhibited LPS-induced inflammatory responses through NF-κB and MAPK in RECs, providing potential therapeutic targets and drugs for the prevention and treatment of SARA.

Physical effectiveness of corn silage fractions stratified with the Penn State Particle Separator for lactating dairy cows.

This study evaluated the physical effectiveness of whole-plant corn silage (CS) particles stratified with the Penn State Particle Separator, composed of 19- and 8-mm-diameter sieves and a pan, for lactating dairy cows. Eight Holstein cows (27.6 ± 2.8 kg/d of milk, 611 ± 74 kg body weight; 152 ± 83 d in milk) were assigned to two 4 × 4 Latin squares (22-d periods, 16-d adaptation), where one square was formed with rumen-cannulated cows. Three CS particle fractions were manually isolated using the 8- and 19-mm diameter sieves and re-ensiled in 200-L drums. The 4 experimental diets were (% dry matter): (1) CON (control): 17% forage neutral detergent fiber (NDF) from CS (basal roughage), 31.5% starch, and 31.9% NDF; (2) PSPan: 17% forage NDF from CS + 9% NDF from CS particles <8 mm, 25.9% starch, and 37.9% NDF; (3) PS8: 17% forage NDF from CS + 9% NDF from CS particles 8 to 19 mm, 25.5% starch, and 38.3% NDF; and (4) PS19: 17% forage NDF from CS + 9% NDF from CS particles >19 mm, 24.9% starch, and 38.8% NDF. Cows fed PS8 had greater dry matter intake and energy-corrected milk yield (22.4 and 26.9 kg/d, respectively) than cows fed CON (20.8 and 24.7 kg/d) and PS19 (21.2 and 24.8 kg/d), but no difference was detected between PSPan (21.6 and 25.8 kg/d) and other treatments. Milk fat concentration was greater for PS8 than CON, with intermediate values for PSPan and PS19. Milk fat yield was greater for cows fed PS8 than CON and PS19, and cows fed PSPan secreted more fat than CON cows but were not different from cows fed the other 2 diets. Cows fed CON had a lower meal frequency than cows fed PSPan, shorter meal and rumination times than PS8, and greater meal size and lower rates of rumination and chewing than the other 3 diets. Total chewing per unit of NDF was higher for PS8 than PSPan, although neither treatment differed from CON or PS19. Cows fed PS19 had higher refusal of feed particles >19 mm than cows fed CON and PSPan. The refusal of dietary NDF and undigested NDF in favor of starch were all greater for PS19 than on the other treatments. Cows fed PS19 had a greater proportion of the swallowed bolus and rumen digesta with particles >19 mm than the other 3 diets. Cows fed CON had the lowest ruminal pH and greatest lactate concentration relative to the other 3 diets. Plasma lipopolysaccharide was higher for cows fed CON and PSPan than for cows fed PS8 and PS19, and serum d-lactate tended to be lower on PSPan than for CON and PS8. In summary, the inclusion of CS fractions in a low-forage fiber diet (CON) reduced signs of ruminal acidosis. Compared with CS NDF <8 and >19 mm, CS NDF with 8- to 19-mm length promoted better rumen health and performance of dairy cows. These results highlight the importance of adjusting CS harvest and formulating dairy diets based on the proportion of particles retained between the 8- and 19-mm sieves.

Acid tolerance of lactate-utilizing bacteria of the order Bacteroidales contributes to prevention of ruminal acidosis in goats adapted to a high-concentrate diet.

The rapid accumulation of organic acids, particularly lactate, has been suggested as the main cause of ruminal acidosis (RA) for ruminants fed high-concentrate diets. Previous research has shown that a gradual shift from low-to high-concentrate diets within 4 to 5 weeks effectively reduces the risk for RA. However, the mechanisms remain unknown. In this study, 20 goats were randomly allocated into four groups (n = 5) and fed with a diet containing a weekly increasing concentrate portion of 20%, 40%, 60%, and 80% over 28 d. At d 7, 14, 21, and 28, one group (named C20, C40, C60, and C80 according to the last concentrate level that they received) was killed and the ruminal microbiome was collected. Ruminal acidosis was not detected in any of the goats during the experiment. Nonetheless, ruminal pH dropped sharply from 6.2 to 5.7 (P < 0.05) when dietary concentrate increased from 40% to 60%. A combined metagenome and metatranscriptome sequencing approach identified that this was linked to a sharp decrease in the abundance and expression of genes encoding nicotinamide adenine dinucleotide (NAD)-dependent lactate dehydrogenase (nLDH), catalyzing the enzymatic conversion of pyruvate to lactate (P < 0.01), whereas the expression of two genes encoding NAD-independent lactate dehydrogenase (iLDH), catalyzing lactate oxidation to pyruvate, showed no significant concomitant change. Abundance and expression alterations for nLDH- and iLDH-encoding genes were attributable to bacteria from Clostridiales and Bacteroidales, respectively. By analyzing the gene profiles of 9 metagenome bins (MAG) with nLDH-encoding genes and 5 MAG with iLDH-encoding genes, we identified primary and secondary active transporters as being the major types of sugar transporter for lactate-producing bacteria (LPB) and lactate-utilizing bacteria (LUB), respectively. Furthermore, more adenosine triphosphate was required for the phosphorylation of sugars to initiate their catabolic pathways in LPB compared to LUB. Thus, the low dependence of sugar transport systems and catabolic pathways on primary energy sources supports the acid tolerance of LUB from Bacteroidales. It favors ruminal lactate utilization during the adaptation of goats to a high-concentrate diet. This finding has valuable implications for the development of measures to prevent RA.

Evaluation of circulating microRNA profiles in blood as potential candidate biomarkers in a subacute ruminal acidosis cow model - a pilot study.

BACKGROUND: Subacute ruminal acidosis (SARA) is a metabolic disorder often observed in high-yielding dairy cows, that are fed diets high in concentrates. We hypothesized that circulating miRNAs in blood of cows could serve as potential candidate biomarkers to detect animals with metabolic dysbalances such as SARA. MicroRNAs (miRNAs) are a class of small non-coding RNAs, serving as regulators of a plethora of molecular processes. To test our hypothesis, we performed a pilot study with non-lactating Holstein-Friesian cows fed a forage diet (FD; 0% concentrate, n = 4) or a high-grain diet (HG; 65% concentrate, n = 4) to induce SARA. Comprehensive profiling of miRNA expression in plasma and leucocytes were performed by next generation sequencing (NGS). The success of our model to induce SARA was evaluated based on ruminal pH and was evidenced by increased time spent with a pH threshold of 5.8 for an average period of 320 min/d. RESULTS: A total of 520 and 730 miRNAs were found in plasma and leucocytes, respectively. From these, 498 miRNAs were shared by both plasma and leucocytes, with 22 miRNAs expressed exclusively in plasma and 232 miRNAs expressed exclusively in leucocytes. Differential expression analysis revealed 10 miRNAs that were up-regulated and 2 that were down-regulated in plasma of cows when fed the HG diet. A total of 63 circulating miRNAs were detected exclusively in the plasma of cows with SARA, indicating that these animals exhibited a higher number and diversity of circulating miRNAs. Considering the total read counts of miRNAs expressed when fed the HG diet, differentially expressed miRNAs ( log2 fold change) and known function, we have identified bta-miR-11982, bta-miR-1388-5p, bta-miR-12034, bta-miR-2285u, and bta-miR-30b-3p as potential candidates for SARA-biomarker in cows by NGS. These were further subjected to validation using small RNA RT-qPCR, confirming the promising role of bta-miR-30b-3p and bta-miR-2285. CONCLUSION: Our data demonstrate that dietary change impacts the release and expression of miRNAs in systemic circulation, which may modulate post-transcriptional gene expression in cows undergoing SARA. Particularly, bta-miR-30b-3p and bta-miR-2285 might serve as promising candidate biomarker predictive for SARA and should be further validated in larger cohorts.

Coordinated response of milk bacterial and metabolic profiles to subacute ruminal acidosis in lactating dairy cows.

BACKGROUND: Bovine milk is an important source of nutrition for human consumption, and its quality is closely associated with the microbiota and metabolites in it. But there is limited knowledge about the milk microbiome and metabolome in cows with subacute ruminal acidosis. METHODS: Eight ruminally cannulated Holstein cows in mid lactation were selected for a 3-week experiment. The cows were randomly allocated into 2 groups, fed either a conventional diet (CON; 40% concentrate; dry matter basis) or a high-concentrate diet (HC; 60% concentrate; dry matter basis). RESULTS: The results showed that there was a decreased milk fat percentage in the HC group compared to the CON group. The amplicon sequencing results indicated that the alpha diversity indices were not affected by the HC feeding. At the phylum level, the milk bacteria were dominated by Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes both in the CON and HC groups. At the genus level, the HC cows displayed an improved proportion of Labrys (P = 0.015) compared with the CON cows. Results of both the principal components analysis and partial least squares of discriminant analysis of milk metabolome revealed that samples of the CON and HC groups clustered separately. A total of 31 differential metabolites were identified between the two groups. Of these, the levels of 11 metabolites decreased (α-linolenic acid, prostaglandin E2, L-lactic acid, L-malic acid, 3-hydroxysebacic acid, succinyladenosine, guanosine, pyridoxal, L-glutamic acid, hippuric acid, and trigonelline), whereas the levels of the other 20 metabolites increased in the HC group with respect to the CON group (P < 0.05). CONCLUSION: These results suggested that subacute ruminal acidosis less impacted the diversity and composition of milk microbiota, but altered the milk metabolic profiles, which led to the decline of the milk quality.

On-farm evaluation of multiparametric models to predict subacute ruminal acidosis in dairy cows.

This research aimed: (i) to evaluate on-farm (FARM data) multiparametric models developed under controlled experiment (INRAE data) and based on non-invasive indicators to detect subacute ruminal acidosis (SARA) in dairy cows. We also aimed to recover high discrimination capacity, if needed, by (ii) building new models with combined INRAE and FARM data; and (iii) enriching the models increasing from 2 to 5 indicators per model. For model enrichment, we focused on indicators determinable on-farm by quick and inexpensive routine analysis. Fifteen commercial dairy farms were selected to cover a wide range of SARA risk. In each farm, four Holstein early-lactating healthy primiparous cows were selected based on their last on-farm recording of milk yield and somatic cell count analysis. Cows were equipped with a reticulo-rumen pH sensor. The pH kinetics were analysed over a subsequent 7-day period. Relative pH indicators were used to classify cows with or without SARA. Milk, blood, faeces, and urine were collected for analysis of the indicators included in the models developed by Villot et al. (2020) on INRAE data that were externally evaluated using FARM data. Then, new models based on the same indicators were developed combining INRAE and FARM data to test whether a possible loss in performance was due to a limited validity domain of model by Villot et al (2020). Finally, the models developed combining INRAE and FARM data were adapted to the on-farm application and enriched by increasing indicators from 2 to 5 per model using linear discriminant analysis and leave-one-out cross-validation. The sensitivities (true-positive rate) in external evaluation on FARM data were substantially lower than those from cross-validation by Villot et al. (2020) (range: 0.1-0.75 vs 0.79-0.96, respectively), and the specificities (true-negative rate) showed a larger range with lower minimum values (range: 0.18-1.0 vs 0.62-0.97, respectively). The sensitivities of new models developed combining INRAE and FARM data ranged from 0.63 to 0.77. Models involving blood cholesterol, ß-hydroxybutyrate, haptoglobin, milk and blood urea, and models involving milk fat/protein ratio, dietary starch proportion, and milk fatty acids had the highest performances, whereas models including sieved faecal residues and urine pH had the lowest. Enriching models to three indicators per model improved sensitivity and specificity, but the inclusion of more indicators was less or not effective. Larger field trials are required to validate our results and to increase variability and validity domain of models.

Effects of High Concentrate-Induced Subacute Ruminal Acidosis Severity on Claw Health in First-Lactation Holstein Cows.

This study aimed to evaluate the effects of diet-induced subacute rumen acidosis (SARA) severity during transition and the early lactation period on claw health in 24 first-lactation Holstein heifers. All heifers were fed a 30% concentrate (in dry matter) close-up ration three weeks before calving, then switched to a high-concentrate ration (60% dry matter), which was fed until the 70th day in milk (DIM) to induce SARA. Thereafter, all cows were fed the same post-SARA ration with around 36% concentrate in dry matter. Hoof trimming was performed before calving (visit 1), at 70 (visit 2) and at 160 DIM (visit 3). All claw lesions were recorded, and a Cow Claw Score (CCS) was calculated for each cow. Locomotion scores (LCS 1-5) were assessed at two-week intervals. Intraruminal sensors for continuous pH measurements were used to determine SARA (pH below 5.8 for more than 330 min in 24 h). The cluster analysis grouped the cows retrospectively into light (≤11%; n = 9), moderate (>11-<30%; n = 7), and severe (>30%; n = 8) SARA groups, based on the percentage of days individual cows experienced SARA. Statistically significant differences were found between SARA groups light and severe in terms of lameness incidence (p = 0.023), but not for LCS and claw lesion prevalence. Further, the analysis of maximum likelihood estimates revealed that for each day experiencing SARA, the likelihood of becoming lame increased by 2.52% (p = 0.0257). A significant increase in white line lesion prevalence was observed between visits 2 and 3 in the severe SARA group. The mean CCS in severe SARA group cows were higher at each visit compared to cows in the other two groups, but without statistical significance. Overall, this is the first study indicating that first-lactation cows fed a similar high-concentrate diet but with a higher severity of SARA tended to have poorer claw health, albeit with only partial statistical evidence.

Characterizing ruminal acidosis risk: A multiherd, multicountry study.

A multicenter observational study was conducted on early lactation Holstein cows (n = 261) from 32 herds from 3 regions (Australia, AU; California, CA; and Canada, CAN) to characterize their risk of acidosis into 3 groups (high, medium, or low) using a discriminant analysis model previously developed. Diets ranged from pasture supplemented with concentrates to total mixed ration (nonfiber carbohydrates = 17 to 47 and neutral detergent fiber = 27 to 58% of dry matter). Rumen fluid samples were collected <3 h after feeding and analyzed for pH, and ammonia, d- and l-lactate, and volatile fatty acid (VFA) concentrations. Eigenvectors were produced using cluster and discriminant analysis from a combination of rumen pH, and ammonia, d-lactate, and individual VFA concentrations and were used to calculate the probability of the risk of ruminal acidosis based on proximity to the centroid of 3 clusters. Bacterial 16S ribosomal DNA sequence data were analyzed to characterize bacteria. Individual cow milk volume, fat, protein, and somatic cell count values were obtained from the closest herd test to the rumen sampling date (median = 1 d before rumen sampling). Mixed model analyses were performed on the markers of rumen fermentation, production characteristics, and the probability of acidosis. A total of 26.1% of the cows were classified as high risk for acidosis, 26.8% as medium risk, and 47.1% as low risk. Acidosis risk differed among regions with AU (37.2%) and CA (39.2%) having similar prevalence of high-risk cows and CAN only 5.2%. The high-risk group had rumen phyla, fermentation, and production characteristics consistent with a model of acidosis that reflected a rapid rate of carbohydrate fermentation. Namely, acetate to propionate ratio (1.98 ± 0.11), concentrations of valerate (2.93 ± 0.14 mM), milk fat to protein ratio (1.11 ± 0.047), and a positive association with abundance of phylum Firmicutes. The medium-risk group contains cows that may be inappetant or that had not eaten recently or were in recovery from acidosis. The low-risk group may represent cattle that are well fed with a stable rumen and a slower rumen fermentation of carbohydrates. The high risk for acidosis group had lower diversity of bacteria than the other groups, whereas CAN had a greater diversity than AU and CA. Rumen fermentation profile, abundance of ruminal bacterial phyla, and production characteristics of early lactation dairy cattle from 3 regions were successfully categorized in 3 different acidosis risk states, with characteristics differing between acidosis risk groups. The prevalence of acidosis risk also differed between regions.

Associations among the genome, rumen metabolome, ruminal bacteria, and milk production in early-lactation Holsteins.

A multicenter observational study to evaluate genome-wide association was conducted in early-lactation Holstein cows (n = 293) from 36 herds in Canada, the USA, and Australia. Phenotypic observations included rumen metabolome, acidosis risk, ruminal bacterial taxa, and milk composition and yield measures. Diets ranged from pasture supplemented with concentrates to total mixed rations (nonfiber carbohydrates = 17 to 47, and neutral detergent fiber = 27 to 58% of dry matter). Rumen samples were collected <3 h after feeding and analyzed for pH, ammonia, d- and l-lactate, volatile fatty acid (VFA) concentrations, and abundance of bacterial phyla and families. Eigenvectors were produced using cluster and discriminant analyses from a combination of pH and ammonia, d-lactate, and VFA concentrations, and were used to estimate the probability of the risk of ruminal acidosis based on proximity to the centroid of 3 clusters, termed high (24.0% of cows), medium (24.2%), and low risk (51.8%) for acidosis. DNA of sufficient quality was successfully extracted from whole blood (218 cows) or hair (65 cows) collected simultaneously with the rumen samples and sequenced using the Geneseek Genomic Profiler Bovine 150K Illumina SNPchip. Genome-wide association used an additive model and linear regression with principal component analysis (PCA) population stratification and a Bonferroni correction for multiple comparisons. Population structure was visualized using PCA plots. Single genomic markers were associated with milk protein percent and the center logged ratio abundance of the phyla Chloroflexi, SR1, and Spirochaetes, and tended to be associated with milk fat yield, rumen acetate, butyrate, and isovalerate concentrations and with the probability of being in the low-risk acidosis group. More than one genomic marker was associated or tended to be associated with rumen isobutyrate and caproate concentrations, and the center log ratio of the phyla Bacteroidetes and Firmicutes and center log ratio of the families Prevotellaceae, BS11, S24-7, Acidaminococcaceae, Carnobacteriaceae, Lactobacillaceae, Leuconostocaceae, and Streptococcaceae. The provisional NTN4 gene, involved in several functions, had pleiotropy with 10 bacterial families, the phyla Bacteroidetes and Firmicutes, and butyrate. The ATP2CA1 gene, involved in the ATPase secretory pathway for Ca2+ transport, overlapped for the families Prevotellaceae, S24-7, and Streptococcaceae, the phylum Bacteroidetes, and isobutyrate. No genomic markers were associated with milk yield, fat percentage, protein yield, total solids, energy-corrected milk, somatic cell count, rumen pH, ammonia, propionate, valerate, total VFA, and d-, l-, or total lactate concentrations, or probability of being in the high- or medium-risk acidosis groups. Genome-wide associations with the rumen metabolome, microbial taxa, and milk composition were present across a wide geographical and management range of herds, suggesting the existence of markers for the rumen environment but not for acidosis susceptibility. The variation in pathogenesis of ruminal acidosis in the small population of cattle in the high risk for acidosis group and the dynamic nature of the rumen as cows cycle through a bout of acidosis may have precluded the identification of markers for acidosis susceptibility. Despite a limited sample size, this study provides evidence of interactions between the mammalian genome, the rumen metabolome, ruminal bacteria, and milk protein percentage.

Relationship between farm management strategies, reticuloruminal pH variations, and risks of subacute ruminal acidosis.

Low reticuloruminal pH (rpH), often observed in subacute ruminal acidosis (SARA), may negatively affect rumen health and animal performance. To investigate the variability of rpH and the prevalence of SARA on commercial farms, we conducted an observational study on 110 early-lactation Holstein cows of different parities from 12 farms selected to cover a broad range of farm management strategies. The rpH of each cow was continuously monitored for 50 d using wireless boluses. To study the effects of animal and farm management characteristics on rpH, we used a multivariable mixed model analysis with the animal and farm as random effects. Automatic milking system and presence of corn silage in the ration were associated with a decrease in rpH of 0.37 and 0.20 pH units, respectively, whereas monensin supplementation was associated with an increase of 0.27 pH units. The rpH increased by 0.15 pH units during the first 60 d in milk. We defined a SARA-positive day as rpH below 5.8 (SARA5.8) or 6.0 (SARA6.0) for at least 300 min for 1 d. Using those definitions, during our study, a total of 38 (35%) and 65 (59%) cows experienced at least one episode of SARA5.8 and SARA6.0, respectively. The proportion of cows with at least one SARA-positive day varied among farms from 0 to 100%. Automatic milking system was associated with an increased risk of SARA5.8 (odds ratio: 10) and SARA6.0 (odds ratio: 11). The use of corn silage was associated with an increased risk of SARA5.8 (odds ratio: 21), whereas the use of monensin was associated with a decreased risk of SARA5.8 (odds ratio: 0.02). Our study shows that rpH is quite variable among farms, but also among animals on the same farm. We also show that multiple animal and farm characteristics are associated with rpH variability and the risk of SARA under commercial conditions.

Blood and milk metabolites of Holstein dairy cattle for the development of objective indicators of a subacute ruminal acidosis.

OBJECTIVE: The purpose of this study was to perform a comparative analysis of metabolite levels in serum and milk obtained from cows fed on different concentrate to forage feed ratios. METHODS: Eight lactating Holstein cows were divided into two groups: a high forage ratio diet (HF; 80% Italian ryegrass and 20% concentrate of daily intake of dry matter) group and a high concentrate diet (HC; 20% Italian ryegrass and 80% concentrate) group. Blood was collected from the jugular vein, and milk was sampled using a milking machine. Metabolite levels in serum and milk were estimated using proton nuclear magnetic resonance and subjected to qualitative and quantitative analyses performed using Chenomx 8.4. For statistical analysis, Student's t-test and multivariate analysis were performed using Metaboanalyst 4.0. RESULTS: In the principal component analysis, a clear distinction between the two groups regarding milk metabolites while serum metabolites were shown in similar. In serum, 95 metabolites were identified, and 13 metabolites (include leucine, lactulose, glucose, betaine, etc.) showed significant differences between the two groups. In milk, 122 metabolites were identified, and 20 metabolites (include urea, carnitine, acetate, butyrate, arabinitol, etc.) showed significant differences. CONCLUSION: Our results show that different concentrate to forage feed ratios impact the metabolite levels in the serum and milk of lactating Holstein cows. A higher number of metabolites in milk, including those associated with milk fat synthesis and the presence of Escherichia coli in the rumen, differed between the two groups compared to that in the serum. The results of this study provide a useful insight into the metabolites associated with different concentrate to forge feed ratios in cows and may aid in the search for potential biomarkers for subacute ruminal acidosis.