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.     

Thiamine supplementation facilitates thiamine transporter expression in the rumen epithelium and attenuates high-grain-induced inflammation in low-yielding dairy cows

An experiment was conducted to uncover the effects of increasing dietary grain levels on expression of thiamine transporters in ruminal epithelium, and to assess the protective effects of thiamine against high-grain-induced inflammation in dairy cows. Six rumen-fistulated, lactating Holstein dairy cows (627 ± 16.9 kg of body weight, 180 ± 6 d in milk; mean ± standard deviation) were randomly assigned to a replicated 3 × 3 Latin square design trial. Three treatments were control (20% dietary starch, dry matter basis), high-grain diet (HG, 33.2% dietary starch, DM basis), and HG diet supplemented with 180 mg of thiamine/kg of dry matter intake. On d 19 and 20 of each period, milk performance was measured. On d 21, ruminal pH, endotoxic lipopolysaccharide (LPS), and thiamine contents in rumen and blood, and plasma inflammatory cytokines were detected; a rumen papillae biopsy was taken on d 21 to determine the gene and protein expression of toll-like receptor 4 (TLR4) signaling pathways. The HG diet decreased ruminal pH (5.93 vs. 6.49), increased milk yield from 17.9 to 20.2 kg/d, and lowered milk fat and protein from 4.28 to 3.83%, and from 3.38 to 3.11%, respectively. The HG feeding reduced thiamine content in rumen (2.89 vs. 8.97 μg/L) and blood (11.66 vs. 17.63 μg/L), and the relative expression value of thiamine transporter-2 (0.37-fold) and mitochondrial thiamine pyrophosphate transporter (0.33-fold) was downregulated by HG feeding. The HG-fed cows exhibited higher endotoxin LPS in rumen fluid (134,380 vs. 11,815 endotoxin units/mL), and higher plasma concentrations of lipopolysaccharide binding protein and pro-inflammatory cytokines when compared with the control group. The gene and protein expression of tumor necrosis factor α (TNFα), IL1B, and IL6 in rumen epithelium increased when cows were fed the HG diet, indicating that local inflammation occurred. The depressions in ruminal pH, milk fat, and protein of HG-fed cows were reversed by thiamine supplementation. Thiamine supplementation increased thiamine contents in rumen and blood, and also upregulated the relative expression of thiamine transporters compared with the HG group. Thiamine supplementation decreased ruminal LPS (49,361 vs. 134,380 endotoxin units/mL) and attenuated the HG-induced inflammation response as indicated by a reduction in plasma IL6, and decreasing gene and protein expression of pro-inflammatory cytokines in rumen epithelium. Western bottling analysis showed that thiamine suppressed the protein expression of TLR4 and the phosphorylation of nuclear factor kappa B (NFκB) unit p65. In conclusion, HG feeding inhibits thiamine transporter expression in ruminal epithelium. Thiamine could attenuate the epithelial inflammation during high-grain feeding, and the protective effects may be due to its ability to suppress TLR4-mediated NFκB signaling pathways.     

Dynamic changes in salivation,salivary composition,and rumen fermentation associated with duration of high-grain feeding in cows

Salivary secretions are essential for the regulation of digestive processes, as well as rumen and cow health. This research evaluated the effects of the duration of high-grain feeding, and of the time relative to a meal, on salivation, saliva properties, feed bolus characteristics, chewing activity, ruminal and reticular volatile fatty acids, as well as salivary and ruminal pH. Nine nonlactating cannulated Holstein cows were sampled at 1 and 23 d after transition to a 65% grain diet (short term and long term, respectively). Both before and after a controlled meal (2.5 kg of dry matter, offered over 4 h), unstimulated saliva was taken orally for composition analysis. Stimulated salivation and feed boli characteristics were evaluated by collection of ingesta from cardia during 30 min. Chewing and ruminal pH were measured during the controlled meal and for a total of 6 h thereafter. Results from unstimulated saliva showed no effect of the duration of high-grain feeding on bicarbonate, phosphate, total proteins, mucins, lysozyme, and buffer capacity, but increased osmolality at the long term. Lysozyme activity did not differ with high-grain feeding duration, but tended to be lower after the meal. In contrast to short-term-fed cows, the long-term-fed cows increased both meal consumption and feed bolus size, but decreased chewing and feed ensalivation (5.2 vs. 4.6 ± 0.50 g of saliva/g of dry matter), and had lower pH of the stimulated saliva (7.00 vs. 6.67 ± 0.076). These cows also had decreased chewing index (66.5 vs. 45.4 min/kg of neutral detergent fiber), and despite the increase in stimulated saliva buffer capacity (0.027 vs. 0.039 ± 0.006), mean ruminal pH decreased (6.31 vs. 6.11 ± 0.065) during ad libitum feeding. Both in the rumen and reticulum, the concentration of total volatile fatty acids was lower and propionate proportion was higher at the long term. Linear regression analyses revealed a positive influence of the flow rates of salivary bicarbonate and phosphate on ruminal pH during the short term. For every 1-mol increment in the flow of bicarbonate or phosphate, ruminal pH increased by 0.062 or 0.439 units, respectively. Overall, salivary buffers are key determinants of ruminal pH regulation, especially during short-term grain feeding. However, in the long term, ruminal pH drop during ad libitum feeding was stronger, and this effect seems to be exacerbated by increased feed bolus size, accompanied by reductions in feed ensalivation, stimulated saliva pH, and chewing index.     

Intramammary infusion of Escherichia coli lipopolysaccharide negatively affects feed intake,chewing, and clinical variables,but some effects are stronger in cows experiencing subacute rumen acidosis

Feeding high-grain diets increases the risk of subacute rumen acidosis (SARA) and adversely affects rumen health. This condition might impair the responsiveness of cows when they are exposed to external infectious stimuli such as lipopolysaccharide (LPS). The main objective of this study was to evaluate various responses to intramammary LPS infusion in healthy dairy cows and those experimentally subjected to SARA. Eighteen early-lactating Simmental cows were subjected to SARA (n = 12) or control (CON; n = 6) feeding conditions. Cows of the control group received a diet containing 40% concentrates (DM basis) throughout the experiment. The intermittent SARA feeding regimen consisted in feeding the cows a ration with 60% concentrate (DM basis) for 32 d, consisting of a first SARA induction for 8 d, switched to the CON diet for 7 d, and re-induction during the last 17 d. On d 30 of the experiment, 6 SARA (SARA-LPS) and 6 CON (CON-LPS) cows were intramammary challenged once with a single dose of 50 μg of LPS from Escherichia coli (O26:B6), whereas the other 6 SARA cows (SARA-PLA) received 10 mL of sterile saline solution as placebo. To confirm the induction of SARA, the reticular pH was continuously monitored via wireless pH probes. The DMI remained unchanged between SARA and CON cows during the feeding experiment, but was reduced in both treatment groups receiving the LPS infusion compared with SARA-PLA, whereby a significant decline was observed for cows of the SARA-LPS treatment (?38%) compared with CON-LPS (?19%). The LPS infusion did not affect the reticuloruminal pH dynamics, but significantly enhanced ruminal temperature and negatively affected chewing behavior. The ruminal temperature increased after the LPS infusion and peaked about 1 h earlier in SARA-LPS cows compared with the cows of the CON-LPS treatment. Moreover, a significant decline in milk yield was found in SARA-LPS compared with CON-LPS following the LPS infusion. Cows receiving LPS had elevated somatic cell counts, protein, and fat contents in milk as well as decreased lactose contents and pH following the LPS infusion, whereby the changes in milk constituents were more pronounced in SARA-LPS than CON-LPS cows. Rectal temperature and pulse rate were highest 6 h after LPS infusion, but rumen contractions were not affected by the LPS infusion. The data suggest that a single intramammary LPS infusion induced fever and negatively affected feed intake, chewing activity, rectal temperature, and milk yield and composition, whereby these effects were more pronounced in SARA cows.     

Population structure of rumen Escherichia coli associated with subacute ruminal acidosis (SARA) in dairy cattle

Previous studies indicated that only subacute ruminal acidosis (SARA), induced by feeding a high-grain diet, is associated with an inflammatory response and increased abundance of Escherichia coli in the rumen. We hypothesized that ruminal E. coli in grain pellet-induced SARA carried virulence factors that potentially contribute to the immune activation during SARA. One hundred twenty-nine E. coli isolates were cultured from the rumens of 8 cows (4 animals per treatment) in which SARA had been nutritionally induced by feeding a high-grain diet (GPI-SARA) or a diet containing alfalfa pellets (API-SARA). The population structure of the E. coli was evaluated with the ABD genotyping system and repetitive sequence-based (rep)-PCR fingerprinting. Twenty-five virulence factors were evaluated with PCR. Escherichia coli numbers were higher in the GPI-SARA treatment than in the API-SARA treatment. The genetic structure of the E. coli was significantly different between SARA challenge models. Isolates from GPI-control (46%), API-control (70%), and API-SARA (53%) were closely related and fell into one cluster, whereas isolates from GPI-SARA (54%) grouped separately. The ABD typing indicated a shift from an A-type E. coli population to a B1-type population only due to GPI-SARA. Of the 25 virulence factors tested, curli fiber genes were highly associated with GPI. Curli fibers were first identified in E. coli mastitis isolates and are potent virulence factors that induce a range of immune responses. Results suggest that under low rumen pH conditions induced by a grain diet, there is a burst in the number of E. coli with virulence genes that can take advantage of these rumen conditions to trigger an inflammatory response.     

Gene function adjustment for carbohydrate metabolism and enrichment of rumen microbiota with antibiotic resistance genes during subacute rumen acidosis induced by a high-grain diet in lactating dairy cows

The high-grain diets fed to ruminants generally alters the structure and function of rumen microbiota, resulting in variations of rumen fermentation patterns and the occurrence of subacute rumen acidosis (SARA). To clarify the microbial mechanism for carbohydrate metabolism during SARA, 8 ruminally cannulated Holstein cows in mid lactation were selected for a 3-wk experiment. The cows were randomly divided into 2 groups, fed either a conventional diet (CON; 40% concentrate; dry matter basis) or a high-grain diet (HG; 60% concentrate; dry matter basis). Compared with the CON diet, the HG diet reduced average daily pH (5.71 vs. 6.13), acetate concentration (72.56 vs. 78.44 mM), acetate ratio (54.81 vs. 65.24%), and the ratio of the concentrations of acetate to propionate (1.87 vs. 3.21) but increased the concentrations of total volatile fatty acids (133.03 vs. 120.22 mM), propionate (41.32 vs. 24.71 mM), and valerate (2.46 vs. 1.68 mM) and the propionate ratio (30.51 vs. 20.47%). Taxonomic analysis indicated that the HG cows had a higher relative abundance of Ruminococcus, Eubacterium, Selenomonas, Ruminobacter, Succinimonas, Methanomicrobium, and Methanocaldococcus accompanied by a lower relative abundance of unclassified Firmicutes, unclassified Bacteroidetes, Bacteroides, Fibrobacter, Alistipes, Candidatus Methanoplasma, Methanomassiliicoccus, and Methanolobus. Carbohydrate-active enzyme annotation suggested that there was enriched abundance of glycosyltransferases (GT) 2, glycoside hydrolase (GH) 13, GH24, carbohydrate-binding module (CBM) 26, GH73, GH25, CBM12, GH23, GT8, CBM50, and GT9 and reduced abundance of GH78, GH31, S-layer homology, GH109, carbohydrate esterase 1, GH3, carbohydrate esterase 10, and GH43 in the HG group. Functional profiling revealed that the HG feeding mainly downregulated the pentose phosphate pathway of carbohydrate catabolism, acetate metabolism, propionate metabolism (succinate pathway), and methane metabolism, whereas it upregulated the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways of glycolysis and the citrate cycle. Additionally, the HG feeding promoted the abundance of various antibiotic resistance genes and antimicrobial resistance gene families. These results elucidated the structure and function adjustment of rumen microbiota for carbohydrate metabolism and summarized the enrichment of rumen antibiotic resistance genes under the HG feeding, which expands our understanding of the mechanism underlying the response of rumen microbiota to SARA in dairy cattle.     

Short-term screening of multiple phytogenic compounds for their potential to modulate chewing behavior,ruminal fermentation profile,and pH in cattle fed grain-rich diets

In cattle, proper rumen functioning and digestion are intimately linked to chewing behavior. Yet, high grain feeding impairs chewing activity, increasing the risk of subacute ruminal acidosis and dysfermentation. This study aimed to screen 9 different phytogenic compounds for their potential to modulate chewing activity, meal size, rumino-reticular short-chain fatty acids (SCFA), and pH during consumption in a first daily meal and shortly thereafter in cattle fed a grain-rich diet. Treatments were control (total mixed ration without phytogenic) or addition of a phytogenic compound at a low or high dose. Phytogenic compounds and doses (all in mg/kg) were angelica root (6.6 and 66), capsaicin (10 and 100), gentian root (6.6 and 66), garlic oil (0.3 and 3), ginger extract (40 and 400), L-menthol (6.7 and 67), mint oil (15.3 and 153), thyme oil (9.4 and 94), and thymol (5 and 50), for the low and high groups, respectively. Before the start of the screening experiment, cows were fed to reach subacute ruminal acidosis conditions, confirmed with the time of ruminal pH <5.8 being 655 ± 148.2 min/d. During the screening experiment, the treatments were offered in a controlled meal (2.5 kg of DM for 4 h) as part of the daily diet with 65% concentrate. Each treatment was tested in 4 of the 9 cannulated Holstein cows using an incomplete Latin square design. Ruminal and reticular fluids were sampled before and after each treatment, and data collected before the meal were used as covariates. Chewing and ruminal pH were monitored during the treatment, followed by 2 h of complete feed restriction, and then 4 h of ad libitum feed intake without phytogenic. Data showed that supplementation of angelica root tended to linearly increase rumination time immediately after the first meal when feed was restricted (27.3, 41.9, and 42.6 ± 5.99 min for control, low and high groups, respectively). Capsaicin increased eating time (43.6, 49.4, and 66.4 ± 4.93 min) during consumption but did not affect ruminal total SCFA or mean ruminal pH. Garlic oil reduced the concentration of reticular total SCFA (75.7, 71.3, and 60.1 mM) and tended to decrease ruminal acetate-to-propionate ratio (2.50, 1.78, and 1.87 ± 0.177) with no effect on ruminal pH. The L-menthol affected reticular total SCFA quadratically (76.1, 64.9, and 81.0 ± 4.22%), and ruminal pH responded quadratically when feed was reintroduced ad libitum (6.0, 6.3, and 6.1 ± 0.07). Mint oil did not affect chewing or total SCFA during consumption, but the low dose increased ruminal pH (6.5, 6.7, and 6.5 ± 0.08). Thyme oil tended to lower the severity of ruminal acidosis. Overall, phytogenic compounds demonstrated distinct dose-dependent effects to beneficially influence chewing behavior, modulate fermentation, and mitigate ruminal acidosis in dairy cows under a high-grain challenge diet.     

Increased feeding frequency increased milk fat yield and may reduce the severity of subacute ruminal acidosis in higher-risk cows

The objectives of this study were to determine whether feeding behavior is different between cows at higher or lower risk for subacute ruminal acidosis (SARA) and whether increasing feeding frequency could be used to reduce the severity of SARA in higher-risk cows. In preliminary studies, 16 ruminally cannulated lactating cows were fed high-grain diets once per day to increase the risk of SARA. After a 17-d diet adaptation, ruminal pH was measured every 30 s over 24 h. Cows were classified as higher-risk (n = 7) or lower-risk (n = 9) for SARA based on an acidosis index (area of pH <5.8/dry matter intake). Feeding behavior was recorded every 5 min over the same 24 h. The 24-h observation period was analyzed in 3 periods of 8 h after feeding. Although there was no significant difference in overall dry matter intake, higher-risk cows spent more time eating in the first 8-h period after feeding than lower-risk cows (186 vs. 153 min) and less time eating in the third 8-h period (19 vs. 43 min). In the primary experiment, 8 ruminally cannulated lactating cows were fed a high-grain diet once per day (1×; 0800 h) or 3 times per day (3×; 0800, 1500, and 2000 h) in a crossover design with 21-d periods (16 d of treatment adaptation and 5 d of data collection). Rumen pH and feeding behavior were measured over 72 h. Behavior data were summarized separately for the 3 periods (0800 to 1500, 1500 to 2200, and 2200 to 0800 h). Four cows were categorized as higher-risk and 4 as lower-risk, based on their acidosis index. The 3× feeding reduced eating time between 0800 and 1500 h (99 vs. 145 min) and increased eating time between 2200 and 0800 h (76 vs. 43 min) for all cows, regardless of category, compared with 1× feeding. For higher-risk cows, 3× feeding reduced the area below pH 5.8 (51 vs. 98 pH × min/d), but it did not affect rumen pH for the lower-risk cows. Milk yield was not different between groups, but 3× feeding increased milk fat yield (1.22 vs. 1.08 kg/d) for all cows, regardless of category, compared to 1× feeding. Our results suggest that cows at higher risk for SARA eat less evenly throughout the day; increasing feeding frequency may reduce the severity of SARA in higher-risk cows and may also increase milk fat yield.     

Active dry Saccharomyces cerevisiae can alleviate the effect of subacute ruminal acidosis in lactating dairy cows

The objective of the study was to determine the effect of active dry Saccharomyces cerevisiae (ADSC) supplementation on dry matter intake, milk yield, milk components, ruminal pH, and microbial community during a dietary regimen that leads to subacute ruminal acidosis (SARA). Sixteen multiparous, rumen-cannulated lactating Holstein cows were randomly assigned to 1 of 2 dietary treatments that included ADSC (Biomate; AB Vista, Marlborough, UK; 8 × 10¹⁰ cfu/head per day) or control. During wk 1 to 6, all cows received a high-forage (HF) diet (77:23, forage:concentrate). Cows were then abruptly switched during wk 7 to a high-grain (HG) diet (49:51, forage:concentrate) and remained on the HG until the end of wk 10. Feed intake and milk yields were recorded daily. Ruminal pH was recorded continuously using an indwelling system for 1 to 2 d per week during the pre-experimental phase, and wk 6, 7, and 10. Ruminal digesta samples were collected at the end of the experiment and analyzed for relative change in microbial communities using real-time quantitative PCR. Cows were considered to have SARA if the duration below pH 5.6 was ≥300 min/d. Ruminal pH during wk 6 (HF plateau) was not different across treatments (15 ± 46 min/d at pH <5.6). The dietary regimen successfully induced SARA during wk 7 (transition from HF to HG diet), and ruminal pH (551 ± 46 min/d at pH <5.6) was not different across treatments. However, cows receiving ADSC had an improved ruminal pH (122 ± 57 vs. 321 ± 53 min/d at pH <5.6) during wk 10 (HG plateau) compared with control. Additionally, cows receiving ADSC had a better dry matter intake (23.3 ± 0.66 vs. 21.6 ± 0.61 kg/d) and 4% fat-corrected milk yield (29.6 ± 1.2 vs. 26.5 ± 1.2 kg/d) than control cows during the HG phase (wk 8 to 10). During HG feeding, cows receiving ADSC had greater total volatile fatty acid and propionate concentrations (175 ± 7.5 vs. 154 ± 7.5 and 117 ± 6.1 vs. 94 ± 5.7 mM for ADSC and control, respectively) and lower acetate:propionate ratio (0.26 ± 0.5 vs. 0.36 ± 0.05 for ADSC and control, respectively). Microbial analyses conducted on samples collected during wk 10 showed that cows supplemented with S. cerevisiae had a 9-fold, 2-fold, 6-fold, 1.3-fold, and 8-fold increase in S. cerevisiae, Fibrobacter succinogenes, Anaerovibrio lipolytica, Ruminococcus albus, and anaerobic fungi, respectively, which suggested an increase in cellulolytic microbes within the rumen. Cows supplemented with ADSC had 2.2-fold reduction in Prevotella albensis, which is a gram-negative bacterium predominant during SARA. Prevotella spp. are suggested to be an important source of lipopolysaccharide responsible for inflammation within the rumen. Cows supplemented with ADSC had a 2.3-fold increase in Streptococcus bovis and a 12-fold reduction in Megasphaera elsdenii. The reduction in M. elsdenii may reflect lower concentration of lactic acid within the rumen for ADSC cows. In conclusion, ADSC supplementation to dairy cows was demonstrated to alleviate the condition of SARA caused by abrupt dietary changes from HF to HG, and can potentially improve rumen function, as indicated by greater numbers of cellulolytic microorganisms within the rumen.     

Epimural bacterial community structure in the rumen of Holstein cows with different responses to a long-term subacute ruminal acidosis diet challenge

Subacute ruminal acidosis (SARA) is a prevalent metabolic disorder in cattle, characterized by intermittent drops in ruminal pH. This study investigated the effect of a gradual adaptation and continuously induced long-term SARA challenge diet on the epimural bacterial community structure in the rumen of cows. Eight rumen-cannulated nonlactating Holstein cows were transitioned over 1 wk from a forage-based baseline feeding diet (grass silage-hay mix) to a SARA challenge diet, which they were fed for 4 wk. The SARA challenge diet consisted of 60% concentrates (dry matter basis) and 40% grass silage-hay mix. Rumen papillae biopsies were taken at the baseline, on the last day of the 1-wk adaptation, and on the last day of the 4-wk SARA challenge period; ruminal pH was measured using wireless sensors. We isolated DNA from papillae samples for 16S rRNA gene amplicon sequencing using Illumina MiSeq. Sequencing results of most abundant key phylotypes were confirmed by quantitative PCR. Although they were fed similar amounts of concentrate, cows responded differently in terms of ruminal pH during the SARA feeding challenge. Cows were therefore classified as responders (n = 4) and nonresponders (n = 4): only responders met the SARA criterion of a ruminal pH drop below 5.8 for longer than 330 min/d. Data showed that Proteobacteria, Firmicutes, and Bacteroidetes were the most abundant phyla, and at genus level, Campylobacter and Kingella showed highest relative abundance, at 15.5 and 7.8%, respectively. Diversity analyses revealed a significant increase of diversity after the 1-wk adaptation but a decrease of diversity and species richness after the 4-wk SARA feeding challenge, although without distinction between responders and nonresponders. At the level of the operational taxonomic unit, we detected diet-specific shifts in epimural community structure, but in the overall epimural bacterial community structure, we found no differences between responders and nonresponders. Correlation analysis revealed significant associations between grain intake and operational taxonomic unit abundance. The study revealed major shifts in the 3 dominating phyla and, most importantly, a loss of diversity in the epimural bacterial communities during a long-term SARA diet challenge, in which 60% concentrate supply for 4 wk was instrumental rather than the magnitude of the drop of ruminal pH below 5.8.     

Models to predict the risk of subacute ruminal acidosis in dairy cows based on dietary and cow factors: A meta-analysis

The present research aimed at developing practical and feasible models to optimize feeding adequacy to maintain desired rumen pH conditions and prevent subacute ruminal acidosis (SARA) in dairy cows. We conducted 2 meta-analyses, one using data from recent published literatures (study 1) to investigate the prediction of SARA based on nutrient components and dietary physical and chemical characteristics, and another using internal data of our 5 different published experiments (study 2) to obtain adjustments based on cow status. The results of study 1 revealed that physically effective neutral detergent fiber inclusive of particles >8 mm (peNDF >8) and dietary starch [% of dry matter (DM)] were sufficient for predicting daily mean ruminal pH {y = 5.960 – (0.00781 × starch) + (0.03743 × peNDF >8) – [0.00061 × (peNDF >8 × peNDF >8)]}. The model for time of pH suppression (<5.8 for ruminal pH or <6.0 for reticular pH, min/d) can be predicted with additionally including DMI (kg/d): 124.7 + (1.7007 × DMI) + (20.9270 × starch) + (0.2959 × peNDF >8) – [0.0437 × (DMI × starch × peNDF >8)]. As a rule of thumb, when taken separately, we propose 15 to 18% peNDF >8 as a safe range for diet formulation to prevent SARA, when starch or NFC levels are within 20 to 25% and 35 to 40% ranges, respectively. At dietary starch content below 20% of DM, grain type was insignificant in affecting ruminal pH. However, increasing dietary starch contents by using corn as the sole grain source could lead to more severe drops of pH compared with using grain mix based on barley and wheat, as underlined by an interaction between starch content and grain type. Data from study 2 emphasized an increased risk of SARA for cows in the first and second lactation with lower mean pH (0.2 units) and double amounts of time at pH <5.8 compared with the cows with ≥3 parities. Given that a lower ruminal pH is expected in these high-risk cows, it is advisable to keep the lower end of recommended starch (20%) and higher peNDF >8 (18%) contents in the diet of these cows. Overall, the present study underlines the possibility of predicting SARA based on dietary factors including peNDF >8 and starch contents, as well as DMI of the cows, which can be practically implemented for optimal diet formulation for dairy cows. With more data available, future studies should attempt to improve the predictions by including additional key dietary and cow factors in the models.     

Modulation of rumen pH by sodium bicarbonate and a blend of different sources of magnesium oxide in lactating dairy cows submitted to a concentrate challenge

With the objective of evaluating the potential effects of sodium bicarbonate or a magnesium-based product on rumen pH and milk performance of dairy cattle exposed to a dietary challenge, 30 lactating Holstein cows (648 ± 67 kg of body weight; 44.4 ± 9.9 kg/d of milk yield; 155 ± 75 d in milk) were blocked by parity (9 primiparous and 21 multiparous) and randomly distributed to 3 treatment groups. One group received a total mixed ration (TMR) that acted as a control (CTR), a second group (SB) received the same TMR but with an additional supplementation of 0.8% of sodium bicarbonate, and a third group (MG) received the same TMR as CTR but an additional supplementation of 0.4% of a magnesium-based product (pHix-Up, Timab, Dinard, France). After 1 wk of exposure to this TMR, all 3 rations were supplemented with 1 kg/d of barley, which was then increased 1 kg/wk until reaching 3 kg/d of barley during wk 4 of the study. Every kilogram of barley replaced 1 kg of forage in the diet. Individual feed intake and behavior were monitored using electronic feed bins. Seven cows per treatment were equipped with an intraruminal bolus that recorded pH every 15 min. As the severity of the barley challenge increased, dry matter intake decreased, but this decrease was more pronounced in SB cows than in MG cows, with an intermediate response for CTR cows. The MG cows produced more milk when challenged with 2 or 3 kg/d of additional barley than when challenged with 1 kg/d, whereas CTR cows produced less milk with the 3 kg/d challenge compared with 1 or 2 kg/d, and the SB cows maintained milk production. Milk fat content decreased with barley challenges, with CTR cows experiencing a more severe decrease than SB cows, which maintained stable butterfat values throughout the study, and MG cows showed a decline in milk fat content only with the 3 kg/d of additional barley. Meal size was also reduced as the severity of barley challenge increased, and this reduction was more modest in MG cows than in SB cows. The number of daily meals consumed by SB and MG cows was more constant than that recorded in CTR cows. Cows on the CTR and SB treatments showed a marked decrease in rumen pH with the 3 kg/d of additional barley, whereas MG cows maintained stable rumen pH during the barley challenges and had greater average rumen pH (5.93 ± 0.04) than CTR cows (5.83 ± 0.04) with the 3 kg/d of additional barley; SB cows showed intermediate values (5.85 ± 0.04). Last, MG cows spent less time (32.3 ± 6.1%) with rumen pH ≤5.8 when exposed to the 3 kg/d of barley challenge than CTR and SB cows (50.7 ± 5.02%). In conclusion, supplementation with MG prevents the decline in dry matter intake and milk production induced by a rumen challenge, whereas supplementation with SB prevents the decay in milk production but does not prevent the decrease in feed intake. These changes were probably due to the ability of the MG treatment to prevent a reduction in rumen pH when challenging cows with 3 kg/d of additional barley in the ration.     

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

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

Subacute ruminal acidosis and total mixed ration preference in lactating dairy cows

Subacute ruminal acidosis (SARA) is a condition where the pH of the rumen becomes abnormally acidic because of increased and altered production of volatile fatty acids. The objective of this experiment was to determine how a SARA challenge affects total mixed ration selection in dairy cows. In this study, 8 multiparous, lactating, ruminally cannulated Holstein cows were given a choice between a long-forage-particle-size diet with slow-fermenting starch (LC) and a short-forage-particle-size diet with fast-fermenting starch in a crossover design. Cows were allowed to adapt to this feeding scheme and were then subjected to a rumen challenge to induce a bout of SARA. The rumen challenge successfully decreased rumen pH and altered rumen volatile fatty acid profiles. Daily average rumen pH decreased from 6.02 to 5.77, and average minimum rumen pH decreased from 5.59 to 5.28. In addition, following the rumen challenge, concentrations of acetate, butyrate, and valerate, and acetate-to-propionate ratio increased. In response to the rumen challenge, intake of LC increased from the baseline level of 18.1% of total daily dry matter intake to 38.3% for that day. During the first recovery day after the rumen challenge, LC intake moderated to 28.0% of total daily dry matter intake. On the second recovery day, LC intake returned to baseline levels at 18.6%. These results indicate that cows are able to alter their diet preference for higher physically effective fiber and slower starch fermentability during a bout of SARA and that they can effectively fully recover from this type of SARA within 72 h when appropriate diets are available.     

Effects on rumen pH and feed intake of a dietary concentrate challenge in cows fed rations containing pH modulators with different neutralizing capacity

Forty-five Holstein lactating cows (41 ± 8.8 kg/d of milk yield, 96 ± 35.6 days in milk, and 607 ± 80.4 kg of body weight) were enrolled in this study to assess the effects of diets supplemented with sodium bicarbonate or a magnesium-based product and their corresponding differences in dietary cation-anion difference (DCAD) on rumen pH, rumen microbial population, and milk performance of dairy cattle exposed to an induced decrease in rumen pH through a dietary challenge. Cows were randomly allocated to 3 total mixed rations (TMR) differing in the type of supplement to modulate rumen pH: (1) control, no supplementation; (2) SB, supplemented with 0.82% of sodium bicarbonate with a neutralizing capacity (NC) of 12 mEq/g; and (3) MG, supplemented with 0.25% of magnesium oxide (pHix-Up, Timab Magnesium) with a NC of 39 mEq/g. Thus, SB and MG rations had, in theory, the same NC. The 3 TMR differed for control, SB, and MG in their DCAD-S (calculated considering Na, K, Cl, and S), which was on average 13.2, 21.2, and 13.7 mEq/100 g, respectively, or DCAD-Mg (calculated accounting for Mg, Ca, and P), which was 31.4, 41.2, and 35.2 mEq/100 g, respectively. The study lasted 63 d, with the first 7 d serving as a baseline, followed by a fortnightly progressive decrease of dietary forage-to-concentrate ratio (FCR) starting at 48:52, then 44:56, then 40:60, and finishing at 36:64. Individual dry matter intake (DMI) was recorded daily. Seven cows per treatment were equipped with electronic rumen boluses to monitor rumen pH. Control and SB cows consumed less dry matter (DM; 23.5 ± 0.31 kg/d) than MG cows (25.1 ± 0.31 kg/d) when fed dietary FCR of 44:56 and 40:60. Energy-corrected milk decreased from 40.8 ± 1.21 to 39.5 ± 1.21 kg/d as dietary FCR decreased, independently of dietary treatments. Rumen pH decreased and the proportion of the day with rumen pH <5.8 increased as dietary FCR decreased, and at low dietary FCR (i.e., 36:64) rumen pH was greater in MG cows than in control and SB cows. Reducing the DCAD-S from 28 to 18 mEq/100 g or the DCAD-Mg from 45 to 39 mEq/kg had no effects on DMI or milk yield. Cows supplemented with ∼62 g/d of magnesium oxide (pHix-Up) maintained a greater rumen pH and consumed more DM than cows supplemented with ∼200 g/d of sodium bicarbonate when fed a diet with low FCR.     

Lipopolysaccharide derived from the digestive tract provokes oxidative stress in the liver of dairy cows fed a high-grain diet

The aims of this study were to measure oxidative stress parameters and to investigate the molecular mechanism triggered by grain-induced subacute ruminal acidosis in mid-lactation cows. Twelve Holstein-Friesian cows with an average weight of 455 ± 28 kg were divided into 2 groups and subjected to 2 diets over 18 wk: either a low-grain (forage-to-concentrate ratio = 6:4) or a high-grain (forage-to-concentrate ratio = 4:6) diet based on dry matter. Being fed a long-term high-grain diet resulted in a significant decrease in rumen pH and a significant increase in ruminal lipopolysaccharide (LPS) at 4 h postfeeding in the morning. The increase was also observed in LPS concentrations in the portal vein, hepatic vein, and jugular vein blood plasma as well as reduced milk yield in a high-grain diet. Cows fed a high-grain diet had lower levels of catalase and glutathione peroxidase (GPx) activity and total antioxidant capacity than cows fed a low-grain diet; however, super oxide dismutase (SOD) activity and malondialdehyde (MDA) levels were higher in both the liver and the plasma of high-grain than in low-grain cows. Positive correlations were observed between plasma LPS versus hepatic MDA, plasma MDA, and hepatic SOD activity, whereas hepatic GPx and plasma GPx were negatively correlated with plasma LPS. The relative mRNA abundances of GPX1 and CAT were significantly lower in the liver of cows fed a high-grain diet than those fed a low-grain diet, whereas SOD1 was significantly higher in cows fed a high-grain diet than cows fed a low-grain diet. The expression levels of Nrf2, NQO1, MT1E, UGT1A1, MGST3, and MT1A were downregulated, whereas NF-kB was upregulated, in cows fed a high-grain diet. Furthermore, nuclear factor E2-related factor 2 (Nrf2) total protein and mRNA levels were significantly lower than in low-grains. Our results demonstrate the relationship between the translocated LPS and the suppression of cellular antioxidant defense capacity, which lead to increased oxidative stress and suggests that the Nrf2-dependent antioxidant response may be affected by higher levels of LPS translocated to the bloodstream.     

Ruminal lipopolysaccharide concentration and inflammatory response during grain-induced subacute ruminal acidosis in dairy cows

The effects of grain-induced subacute ruminal acidosis (SARA) in lactating dairy cows on free ruminal lipopolysaccharide (LPS) and indicators of inflammation were determined. Four mid lactation dairy cows were divided into 2 groups of 2 cows and used in a repeated switchover design. During each period, SARA was induced in 2 animals for 5 subsequent days by replacing 25% of their total mixed ration (dry matter basis) with a concentrate made of 50% wheat and 50% barley. The other 2 cows acted as controls and were fed a total mixed ration diet in which 44% of dry matter was concentrate. On average, inducing SARA did not affect milk composition, increased the duration of rumen pH below 5.6 from 187 to 309 min/d, and increased free ruminal LPS concentration from 24,547 endotoxin units (EU)/mL to 128,825 EU/mL. Averaged across treatments, milk fat yield and milk protein yield were 0.66 and 1.00 kg/d, respectively. Rumen pH and milk fat data suggest that control cows also experienced ruminal acidosis, albeit a milder form of this disease than SARA cows. Serum LPS concentration in both control and SARA cows was less than the detection limit of <0.01 EU/mL for the assay. Induction of SARA elevated serum amyloid A concentration from 286.8 to 498.8 μg/mL, but did not affect other markers of inflammation including haptoglobin, fibrinogen, serum copper, or white blood cells. These results suggest that grain-induced SARA in mid lactation dairy cows increases the lysis of gram-negative bacteria and activates an inflammatory response.     

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

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

Adaptive responses in short-chain fatty acid absorption,gene expression,and bacterial community of the bovine rumen epithelium recovered from a continuous or transient high-grain feeding

The feeding of high-grain diets to dairy cows commonly results in lowered pH and ruminal dysbiosis, characterized by changes in absorption dynamics of short-chain fatty acids (SCFA) across the reticuloruminal wall, epithelial function, and the epithelial bacteria community structure. Therefore, the present study evaluated the effect of high-grain feeding persistence on the absorption kinetics of reticuloruminal SCFA, gene expression in the rumen epithelium, and the associated shifts in the epithelial bacteria in cows recovering from either a long-term continuous high-grain feeding model or a long-term transient high-grain feeding model. In a crossover study design, 8 nonlactating Holstein cows were fed 60% concentrate either continuously for 4 wk (continuous) or with a 1-wk break in the second week of the high-grain feeding (transient). After the high-grain feeding, all animals were fed a diet of 100% forage (recovery) for an additional 8 wk. Rumen papilla biopsies and SCFA absorption measurements were taken at the start of the trial (baseline), after the 4-wk high-grain feeding (49 d), after 2-wk recovery forage feeding (63 d), and after 8-wk recovery forage (105 d). Absorption of SCFA was determined in vivo using the washed and isolated reticulorumen technique. Rumen papillae biopsies were used for adherent bacterial DNA and host RNA extraction. The epithelial bacteria were determined using Illumina MiSeq (Microsynth AG, Balgach, Switzerland) sequencing of the 16S rRNA gene. No significant effects of the high-grain feeding model were seen for bacterial diversity. However, bacterial diversity increased with time spent in the recovery forage feeding period regardless of feeding model. The relative abundance of Acidobacteria phyla and Acetivibrio spp. increased when animals were fed a transient high-grain feeding model. A trend toward increased CLDN4 expression was observed in the continuous model. Furthermore, there were interactions between feeding model and sampling day for gene targets CD14, DRA, NHE2, NHE3, and MCT2. When comparing length of recovery, in the continuous model increased relative absorption of SCFA was sustained at 63 d but dropped to baseline measurements at 105 d. A similar pattern was found with the transient model but it did not reach significance. The only gene target that was found to significantly correlate to relative absorption of SCFA was DRA (correlation coefficient ≤ −0.41). Whereas, genera Alkalibaculum, Anaerorhabdus, Coprococcus, and Dethiobacter all showed positive correlations to gene targets for pH regulation (NHE2 and NHE3) and SCFA uptake (MCT1) but negative correlations to SCFA absorption. We conclude that while the rumen absorption and epithelial bacteria were able to recover to baseline levels after 8 wk of forage feeding, the time needed for re-establishment of homeostasis in host gene expression is longer, especially when high-grain feeding is interrupted.     

Invited review: Practical feeding management recommendations to mitigate the risk of subacute ruminal acidosis in dairy cattle

Rumen health is of vital importance in ensuring healthy and efficient dairy cattle production. Current feeding programs for cattle recommend concentrate-rich diets to meet the high nutritional needs of cows during lactation and enhance cost-efficiency. These diets, however, can impair rumen health. The term “subacute ruminal acidosis” (SARA) is often used as a synonym for poor rumen health. In this review, we first describe the physiological demands of cattle for dietary physically effective fiber. We also provide background information on the importance of enhancing salivary secretions and short-chain fatty acid absorption across the stratified squamous epithelium of the rumen; thus, preventing the disruption of the ruminal acid–base balance, a process that paves the way for acidification of the rumen. On-farm evaluation of dietary fiber adequacy is challenging for both nutritionists and veterinarians; therefore, this review provides practical recommendations on how to evaluate the physical effectiveness of the diet based on differences in particle size distribution, fiber content, and the type of concentrate fed, both when the latter is part of total mixed ration and when it is supplemented in partial mixed rations. Besides considering the absolute amount of physically effective fiber and starch types in the diet, we highlight the role of several feeding management factors that affect rumen health and should be considered to control and mitigate SARA. Most importantly, transitional feeding to ensure gradual adaptation of the ruminal epithelium and microbiota; monitoring and careful management of particle size distribution; controlling feed sorting, meal size, and meal frequency; and paying special attention to primiparous cows are some of the feeding management tools that can help in sustaining rumen health in high-producing dairy herds. Supplementation of feed additives including yeast products, phytogenic compounds, and buffers may help attenuate SARA, especially during stress periods when the risk of a deficiency of physically effective fiber in the diet is high, such as during early lactation. However, the usage of feed additives cannot fully compensate for suboptimal feeding management.