Technical note: Changes in rumen mucosa thickness measured by transabdominal ultrasound as a noninvasive method to diagnose subacute rumen acidosis in dairy cows

Feeding high-grain diets leads to the release and accumulation of short-chain fatty acids in the rumen. The subsequent prolonged decline in ruminal pH can lead to subacute ruminal acidosis (SARA). Accumulation of short-chain fatty acids can cause proliferation of rumen papillae to increase absorption surface, subsequently leading to a thickening of the rumen mucosa. The aim of this study was to evaluate the appropriateness of continuous measurements of the rumen mucosa thickness (RMT) as a diagnostic tool for SARA in dairy cows compared with continuous measurements of ruminal pH. The study used 6 lactating Simmental cows switched from a moderate-grain (MG) diet with 40% concentrate (dry matter basis) for 1 wk to a high-grain (HG) diet with 60% concentrate (dry matter basis) for 4 wk. Reticuloruminal pH was recorded with indwelling sensors throughout the trial. Rumen mucosa thickness was measured by transabdominal ultrasound at 4 d during the MG diet and 23 d during the HG diet. Mean RMT increased from 4.7 ± 0.19 mm in the MG diet to 5.3 ± 0.17 mm in the HG diet, whereas daily mean reticular pH decreased from 6.8 ± 0.01 in the MG diet to 6.5 ± 0.01 in the HG diet. Older cows (>3 lactations) had increased RMT, associated with higher reticular pH throughout the experiment. The higher RMT and pH level in older cows underlines their lesser susceptibility to SARA during high-grain feeding. In conclusion, RMT can successfully be measured using linear ultrasound probes, commonly used by veterinary practitioners as rectal probes. By combining noninvasive RMT measurements with the lactation number of the individual cows in a herd, this study suggests that RMT is a viable option for diagnosing SARA. Further research, using a larger number of cows with different lactations numbers, is needed to establish a cut-off RMT indicating the risk of SARA.     

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.     

Thiamine ameliorates metabolic disorders induced by a long-term high-concentrate diet and promotes rumen epithelial development in goats

Data indicate that dietary thiamine supplementation can partly alleviate rumen epithelium inflammation and barrier function in goats fed a high-concentrate diet. The current work aimed to explore whether thiamine promotes rumen epithelium development by regulating carbohydrate metabolism during a long period of feeding high levels of concentrate. For the experiment, 24 female Boer goats (35.62 ± 2.4 kg of body weight) in parity 1 or 2 were allocated to 3 groups (8 goats per replicate) receiving a low-concentrate diet (concentrate:forage 30:70), a high-concentrate diet (HC; concentrate:forage 70:30), or a high-concentrate diet (concentrate:forage 70:30) supplemented with 200 mg of thiamine/kg of dry matter intake (HCT; concentrate:forage 70:30). On the last day of 12 wk, rumen fluid and blood samples were collected to measure ruminal parameters, endotoxin lipopolysaccharide, and blood inflammatory cytokines. Goats were slaughtered to collect ruminal tissue to determine differential metabolites, enzyme activities, and gene expression. Liquid chromatography–tandem mass spectrometry analysis revealed that the HCT group had significantly increased concentrations of d-glucose 6-phosphate, d-fructose 6-phosphate, glyceraldehyde 3-phosphate, thiamine pyrophosphate, oxaloacetate, acetyl-CoA, succinyl-CoA, sedoheptulose 7-phosphate, ribose 5-phosphate, and NADPH compared with the HC group. The pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and transketolase enzyme activities in the rumen epithelium of the HCT group were higher than those in the HC group. The plasma total antioxidant capacity values for the HCT group were greater than those for the HC group. The rumen epithelium ATP content in the HCT group was higher than that in the HC group. Compared with the HCT group, the HC group had a lower mRNA abundance of CCND1, CCNA2, CDK2, CDK4, CDK6, BCL2, PI3K, and AKT1. Taken together, the results suggest that dietary thiamine supplementation could ameliorate disorders in the tricarboxylic acid cycle and the pentose phosphate pathway induced by a long-term high-concentrate diet and could promote rumen epithelial growth.     

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.     

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.     

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.     

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.     

Effects of extrusion of grain and feeding frequency on rumen fermentation, nutrient digestibility, and milk yield and composition in dairy cows.

The effect of corn extrusion and feeding frequency on ruminal and postruminal digestibility and milk yield was studied in cows fed a high concentrate diet. Four Israeli Holstein cows fitted with rumen and abomasal cannulas were used. The experiment was arranged as a 2 x 2 factorial design, with two diets and two feeding frequencies (two or four meals per day). One diet contained 40% ground corn. In the second diet, half of the ground corn was replaced with extruded corn. Feeding cows the extruded versus ground corn diet decreased ruminal ammonia N and plasma urea N concentrations, increased postruminal digestibility of nonstructural carbohydrates, reduced dry matter intake, decreased yield of milk and milk components, and increased efficiency of milk energy and milk protein synthesis. The inclusion of extruded corn in the diet did not affect ruminal volatile fatty acid. Increasing the feeding frequency reduced the diurnal variation in ruminal pH, ruminal ammonia, and plasma urea, and increased dry matter intake--considerably more in the cows fed ground versus extruded corn--and improved postruminal organic matter, nonstructural carbohydrate, and crude protein digestibility. Total tract digestibility of organic matter and crude protein and milk yield and composition were also increased when cows were fed four versus two meals. Concurrent with the feeding frequency and grain processing effect, an increase in rumen-undegradable protein flow was related to increased digestion of nonstructural carbohydrate postruminally (r = 0.54). We concluded that for cows fed high-starch diets more frequent meals are useful for improving postruminal digestibility and milk yield and composition.     

Effect of supplemental yeast culture and dietary starch content on rumen fermentation and digestion in dairy cows

The objectives of this experiment were to evaluate the effect of feeding a culture of Saccharomyces cerevisiae on rumen metabolism and digestibility when cows are fed diets varying in starch content. Four lactating Holstein cows were assigned to a 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments. Treatments were low starch (LS; 23% of diet DM) and no yeast culture (YC; LS-control), LS and 15 g of YC/d (LS-YC), high starch (HS; 29% of diet DM) and no YC (HS-control), and HS and 15 g of YC/d (HS-YC). Periods lasted 28 d, with the last 9 d for data collection. Days 20 to 24 were used to determine production, nutrient flow, and digestibility. On d 25, 3 kg of corn grain DM was placed in the rumen 1 h before the morning feeding, and yields of milk and milk components were measured after the challenge. Blood was sampled −1, 3, 7, and 11 h relative to the morning feeding on d 24 and 25. Rumen pH was measured continuously on d 24 and 25. Rumen papillae were collected on d 24 and 28 to quantify mRNA expression of select genes. Supplementing YC increased yields of milk (26.3 vs. 29.6 kg/d), energy-corrected milk (ECM; 26.5 vs. 30.3 kg/d), fat (0.94 vs. 1.08 kg/d), true protein (0.84 vs. 0.96 kg/d), and ECM/dry matter intake (1.15 vs. 1.30) compared with the control but did not affect dry matter intake (22.6 vs. 22.9 kg/d). Cows fed HS had increased milk true protein percentage (3.18 vs. 3.31%) and yield (0.87 vs. 0.94 kg/d) compared with cows fed LS. Feeding HS-YC increased the proportion of dietary N incorporated into milk true protein from 24.9% in the other 3 treatments to 29.6%. Feeding HS increased the concentration of propionate (21.7 vs. 32.3 mM) and reduced that of NH₃-N (8.3 vs. 6.7 mg/dL) in rumen fluid compared with the control, and combining HS with YC in HS-YC tended to increase microbial N synthesis compared with LS-YC (275 vs. 322 g/d). Supplementing YC to cows fed HS reduced plasma haptoglobin and rumen lactate concentrations, increased mean rumen pH, reduced the time with pH <6.0, and prevented the decrease in rumen neutral detergent fiber digestion caused by HS. Cows fed HS had less total-tract digestion of organic matter (73.9 vs. 72.4%) because of reduced acid detergent fiber (57.6 vs. 51.7%) and neutral detergent fiber (60.9 vs. 56.7%) digestibility. Production performance after the challenge was similar to that before the challenge, and YC improved yield of ECM. After the challenge, supplementing YC tended to reduce rumen lactate concentration compared with the control and reduced haptoglobin in cows fed HS. Feeding HS but not YC increased expression in rumen papillae of genes for receptors (FFAR2 and FFAR3) and transporter (SLC16A3) of short-chain fatty acids but did not affect genes involved in transport of Na⁺/H⁺ or water or in inflammatory response. Supplementing YC to dairy cows improved lactation performance in diets containing low or high starch, and mechanisms might be partially attributed to improvements in rumen pH, digestion of fiber, microbial N synthesis, and reduction in acute phase response.     

Assessment of rumen bacteria in dairy cows with varied milk protein yield

The present study was conducted to assess rumen bacteria in lactating cows with different milk protein yield, aiming to understand the role of rumen bacteria in this trait. Cows with high milk protein yield (high milk yield and high milk protein content, HH; n = 20) and low milk protein yield (low milk yield and low milk protein content, LL; n = 20) were selected from 374 mid-lactation Holstein dairy cows fed a high-grain diet. Measurement of the rumen fermentation products showed that the concentrations of ruminal total volatile fatty acids, propionate, butyrate, and valerate and the proportion of isobutyrate were higher in the HH cows than in the LL cows. Amplicon sequencing analysis of the rumen bacterial community revealed that the richness (Chao 1 index) of rumen microbiota was higher in the LL cows than in the HH cows. Among the 10 predominant bacterial phyla (relative abundance being >0.10%, present in >60% of animals within each group), the relative abundance of Proteobacteria was 1.36-fold higher in the HH cows than in the LL cows. At the genus level, the relative abundance of Succinivibrio was significantly higher and that of Clostridium tended to be higher in the LL cows than in the HH cows. Sharpea was 2.28-fold enriched in the HH cows compared with the LL cows. Different relationships between the relative abundances of rumen microbial taxa and volatile fatty acid concentrations were observed in the HH and the LL animals, respectively. Succinivibrio and Prevotella were positively correlated with acetate, propionate, and valerate in the LL cows, whereas Sharpea was positively correlated with propionate and valerate concentrations in the HH cows. Collectively, our results revealed that rumen bacterial richness and the relative abundances of several bacterial taxa significantly differed between dairy cows with high and low milk protein yields, suggesting the potential roles of rumen microbiota contributing to milk protein yield in dairy cows.     

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.     

Effects of lactic acid bacteria in a silage inoculant on ruminal nutrient digestibility,nitrogen metabolism,and lactation performance of high-producing dairy cows

Silage treated with lactic acid bacteria inoculants has been reported to increase ruminal microbial biomass when tested in vitro. Therefore, we tested if alfalfa silage inoculated with Lactobacillus plantarum MTD-1 would improve ruminal N metabolism and increase milk production in high-producing dairy cows. Twenty-eight early lactation Holstein cows (8 ruminally cannulated) were blocked by DIM and milk production; animals were used in a double crossover design consisting of four 28-d periods. Animals in each block were randomly assigned to 2 treatments: a diet containing uninoculated alfalfa silage (control) and a diet containing alfalfa silage inoculated with L. plantarum MTD-1 (LP). Diets were formulated to contain 50% of alfalfa silage, 16% crude protein, and 25% neutral detergent fiber (dry matter basis). Milk production and dry matter intake were recorded in the last 14 d of each period. Milk samples were collected twice at both daily milkings on d 20, 21, 27, and 28 of each period. On d 22, omasal samples were collected from the cannulated animals over a period of 3 d to quantify ruminal digestibility and nutrient flows. Data were analyzed using mixed models of SAS 9.4 (SAS Institute). Compared to the control, cows receiving the LP treatment had greater milk production (40.4 vs. 39.6 kg/d) and lower milk urea nitrogen concentration (11.6 vs. 12.7 mg/dL), despite minor changes in energy-corrected milk. Milk lactose concentration was greater in the milk produced by cows fed the LP treatment, which reflected a tendency for increased milk lactose yield. Although milk true protein concentration was lower for cows in the LP treatment, milk true protein yield was the same on both control and LP treatments. Improvements in milk production of animals under the LP treatment were associated with greater organic matter truly digested in the rumen, especially ruminal neutral detergent fiber digestion. Minor changes were observed in total omasal microbial nonammonia N flow in cows receiving the LP treatment. Therefore, alfalfa silage treated with L. plantarum MTD-1 may improve ruminal fermentation and milk production; however, because of a lack of response in ruminal N metabolism, these changes did not result in greater energy-corrected milk in high-producing dairy cows.     

Rumen epithelial adaptation to ruminal acidosis in lactating cattle involves the coordinated expression of insulin-like growth factor-binding proteins and a cholesterolgenic enzyme

The objective of this study was to characterize the mRNA expression of metabolic and proliferative genes in the rumen epithelium during ruminal acidosis. To meet our objectives, 16 rumen-fistulated, lactating Holstein dairy cattle (618±35 kg of body weight, 221±32 d in milk) were used in a randomized complete block design. All cattle were fed a high-forage diet (HF; 88.9% of dry matter) for 5 wk before the experiment. After the baseline week (wk 0), half of the cattle were randomly assigned and transitioned to a high-concentrate diet (HC; 62.2% of dry matter) which was fed for 3 wk (wk 1, 2, and 3). For the last 48 h of each week, continuous ruminal pH, short-chain fatty acids, and plasma β-hydroxybutyrate were assessed, followed by a rumen papillae biopsy. Milk production was higher in HC cattle compared with HF during wk 1, 2, and 3 (17.4±0.5 vs. 23.4±0.9 kg/d, respectively); however, the mean ruminal pH was decreased (5.75±0.03 vs. 6.30±0.02). The HC cattle spent more time below pH 5.6 (594±54 vs. 3±3 min/d) and displayed greater concentrations of ruminal butyrate (15.8±0.9 vs. 10.2±0.4 mmol) and plasma β-hydroxybutyrate (1,036±63 vs. 778±20 μM) compared with the HF cattle. The mRNA expression of genes involved in ketogenesis (HMGCS2 and PPARA) and short-chain fatty acid transport (MCT1) was unchanged by treatment. However, a downregulation in HMGCS1 (0.72±0.09), one of the cholesterol biosynthesis genes, was observed in HC cattle during wk 1 of the grain challenge. In addition, the relative mRNA expression value of insulin-like growth factor-binding protein 3 was lower (0.78±0.06), whereas insulin-like growth factor-binding protein 5 was higher (1.79±0.15) in HC compared with HF cattle. These results suggest that grain-induced ruminal acidosis alters the mRNA expression of IGF-binding proteins and a cholesterolgenic enzyme in the rumen epithelium of lactating dairy cattle.     

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.     

A grain-based subacute ruminal acidosis challenge causes translocation of lipopolysaccharide and triggers inflammation

The effects of a grain-based subacute ruminal acidosis (SARA) challenge on translocation of lipopolysaccharide (LPS) into the peripheral circulation, acute phase proteins in blood and milk, feed intake, milk production and composition, and blood metabolites were determined in 8 lactating Holstein cows. Between wk 1 and 5 of 2 successive 6-wk periods, cows received a total mixed ration ad libitum with a forage to concentrate (F:C) ratio of 50:50. In wk 6 of both periods, the SARA challenge was conducted by replacing 21% of the dry matter of the total mixed ration with pellets containing 50% wheat and 50% barley. Rumen pH was monitored continuously using indwelling pH probes in 4 rumen cannulated cows. Rumen fluid samples were collected 15 min before feed delivery and at 2, 4, 6, 12, 14, 16, 18, and 24 h after feed delivery for 2 d during wk 5 (control) and wk 6 (SARA). Peripheral blood samples were collected using jugular catheters 15 min before feeding and at 6 and 12 h after feeding at the same days of the rumen fluid collections. The SARA challenge significantly reduced average daily pH from 6.17 to 5.97 and increased the duration of rumen pH below pH 5.6 from 118 to 279 min/d. The challenge reduced dry matter intake (16.5 vs. 19 kg/d), milk yield (28.3 vs. 31.6 kg/d), and milk fat (2.93 vs. 3.30%, 0.85 vs. 0.97 kg/d), and tended to increase milk protein percentage (3.42 vs. 3.29%), without affecting milk protein yield (1.00 vs. 0.98 kg/d). The challenge also increased the concentration of free LPS in rumen fluid from 28,184 to 107,152 endotoxin units (EU)/mL. This was accompanied by an increase in LPS in peripheral blood plasma (0.52 vs. <0.05 EU/mL) with a peak at 12 h after feeding (0.81 EU/mL). Concentrations of the acute phase proteins serum amyloid A, haptoglobin, and LPS-binding protein (LBP) in peripheral blood as well as LBP concentration in milk increased (438.5 vs. 167.4, 475.6 vs. 0, 53.1 vs. 18.2, and 6.94 vs. 3.02 μg/mL, respectively) during SARA. The increase in LBP in combination with the increase in LPS in peripheral blood provides additional evidence of translocation of LPS. Results suggest that the grain-based SARA challenge resulted in translocation of LPS into the peripheral circulation, and that this translocation triggered a systemic inflammatory response.     

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.     

Effect of grains differing in expected ruminal fermentability on the productivity of lactating dairy cows

The objective of the study was to evaluate the effect of barley and corn grains differing in expected fermentability in the rumen on dry matter intake (DMI) and productivity of lactating dairy cows. Twenty-two multiparous and 9 primiparous lactating Holstein cows (94 ± 29 d in milk; mean ± SD) were used in a 3 × 3 Latin square design with 21-d periods. Experimental diets contained approximately 40% of dietary dry matter as steam-rolled barley, using a lot of cultivar Dillon or cultivar Xena, or a corn mixture (CM) containing 87.5% dry ground corn, 11.4% beet pulp, and 1.1% urea (dry matter basis). Starch concentration of the grain sources was 50.0, 58.7, and 60.4% and in vitro 6-h starch digestibility was 73.5, 78.0, and 71.0%, respectively, for Dillon, Xena, and CM. All diets were formulated to contain 19.4% crude protein and 25.3% forage neutral detergent fiber. Dry matter intake (23.6 vs. 21.6 kg/d) and yields of milk (40.4 vs. 37.4 kg/d), milk protein (1.20 vs. 1.12 kg/d), and milk lactose (1.85 vs. 1.74 kg/d) were higher for cows fed CM than for cows fed barley. Although DMI was similar for cows fed Xena and Dillon (21.9 vs. 21.4 kg/d), cows fed Xena had higher yields of milk (38.5 vs. 36.2 kg/d), milk protein (1.18 vs. 1.07 kg/d), and milk lactose (1.80 vs. 1.69 kg/d) than cows fed Dillon. However, milk fat concentration tended to be higher (3.47 vs. 3.23%) for cows fed Dillon than Xena. Plasma glucose and nonesterified fatty acid concentrations were not affected by treatment, but plasma insulin concentration was higher for cows fed Xena compared with those fed Dillon (8.50 vs. 5.91 μIU/mL). Greater milk production for cows fed CM can be attributed to greater DMI. Feeding barley that was lower in starch concentration and ruminal starch fermentability (Dillon) did not increase DMI compared with feeding barley that was higher in starch concentration and ruminal starch fermentability (Xena). Reducing ruminal starch degradation of barley grain may not improve the productivity of lactating dairy cows.     

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.