A 2-year study reveals implications of feeding management and exposure to mycotoxins on udder health, performance, and fertility in dairy herds.

We recently reported the ubiquitous occurrence of mycotoxins and their secondary metabolites in dairy rations and a substantial variation in the feeding management among Austrian dairy farms. The present study aimed to characterize to which extent these factors contribute to the fertility, udder health traits, and performance of dairy herds. During 2019 and 2020, we surveyed 100 dairy farms, visiting each farm 2 times and collecting data and feed samples. Data collection involved information on the main feed ingredients, nutrient composition, and the levels of mycotoxin and other metabolites in the diet. The annual fertility and milk data of the herds were obtained from the national reporting agency. Calving interval was the target criterion for fertility performance, whereas the percentage of primiparous and multiparous cows in the herd with somatic cell counts above 200,000 cells/mL was the criterion for impaired udder health. For each criterion, herds were classified into 3 groups: high/long, mid, and low/short, with the cut-off corresponding to the <25th and >75th percentiles and the rest of the data, respectively. Accordingly, for the calving interval, the cut-offs for the long and short groups were ≥400 and ≤380 d, for the udder health in primiparous cows were ≥20% and ≤8% of the herd, and for the udder health in multiparous cows were ≥35% and ≤20% of the herd, respectively. Quantitative approaches were further performed to define potential risk factors in the herds. The high somatic cell count group had higher dietary exposure to enniatins (2.8 vs. 1.62 mg/cow per d), deoxynivalenol (4.91 vs. 2.3 mg/cow per d), culmorin (9.48 vs. 5.72 mg/cow per d), beauvericin (0.32 vs. 0.18 mg/cow per d), and siccanol (13.3 vs. 5.15 mg/cow per d), and total Fusarium metabolites (42.8 vs. 23.2 mg/cow per d) and used more corn silage in the ration (26.9% vs. 17.3% diet DM) compared with the low counterparts. Beauvericin was the most substantial contributing variable among the Fusarium metabolites, as indicated by logistic regression and modeling analyses. Logistic analysis indicated that herds with high proportions of cows with milk fat-to-protein ratio >1.5 had an increased odds for a longer calving interval, which was found to be significant for primiparous cows (odds ratio = 5.5, 95% confidence interval = 1.65-21.7). As well, herds with high proportions of multiparous cows showing levels of milk urea nitrogen >30 mg/dL had an increased odds for longer calving intervals (odds ratio = 2.96, 95% confidence interval = 1.22-7.87). In conclusion, the present findings suggest that dietary contamination of Fusarium mycotoxins (especially emerging ones), likely due to increased use of corn silage in the diet, seems to be a risk factor for impairing the udder health of primiparous cows. Mismatching dietary energy and protein supply of multiparous cows contributed to reduced herd fertility performance.

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

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

Replacing concentrates with a high-quality hay in the starter feed of dairy calves: II. Effects on the development of chewing and gut fermentation, and selected systemic health variables.

Early development of the rumen, rumination, and fermentation is highly important in dairy calves. Yet, common rearing practices with feeding of concentrate-rich starters may jeopardize them because of lacking physically effective fiber (peNDF). The main objective of this study was to establish the influence of the composition of the calf starter feed (only forage with 2 different qualities or concentrate-rich starter diet) on chewing behavior, rumen development, rumen and hindgut fermentation, and selected systemic health and stress variables of dairy calves. The experiment was carried out with 40 newborn Holstein-Friesian calves, randomly assigned to 4 different solid feed treatments: MQH = 100% medium-quality hay (9.4 MJ metabolizable energy, 149 g of crude protein, and 522 g of neutral detergent fiber/kg of dry matter); HQH = 100% high-quality hay (11.2 MJ of metabolizable energy, 210 g of crude protein, 455 g of neutral detergent fiber/kg of dry matter); MQH+C = 30% MQH + 70% starter concentrate; HQH+C = 30% HQH + 70% starter concentrate). All calves were up to 14 wk in the trial and received acidified whole milk ad libitum in the first 4 wk of life, thereafter in reduced quantity until weaning on 12 wk of age. Water and the solid feed treatments were available ad libitum throughout the trial. Chewing activity was measured in wk 4, 6, 10, and 12 using RumiWatch halters. Until wk 3, rumen fluid, feces and blood were sampled weekly, thereafter every 2 wk. Rumen mucosal thickness (RMT) was measured on the same days with rumen fluid samples. Data showed that calves fed the HQH diet consumed more peNDF and this was associated with longer rumination time (591 min/d) and more ruminating boli (709 boli/d) than calves fed concentrate-rich diets (MQH+C: 430 min/d, 518 boli/d; HQH+C: 430 min/d, 541 boli/d), whereas the MQH group was intermediate (539 min/d, 644 boli/d). Ruminal and fecal pH were higher in calves fed only hay (especially MQH) compared with calves with concentrate supplementation. In both hay-fed groups, ruminal and fecal short-chain fatty acids were shifted toward acetate, whereas only the HQH diet increased the butyrate proportion in the ruminal short-chain fatty acids profile. Ruminal ammonia concentration was at a high level only in the first 3 wk and decreased thereafter. Feeding HQH tended to increase ruminal ammonia, likely because of its high crude protein content and ruminal degradability as well as lower assimilation from rumen microbes. The RMT similarly, though nonlinearly, increased in all groups over the course of the experiment. When using RMT as an indicator of rumen development in dairy calves in the practice, our data suggest an RMT of 1.7 mm and >2 mm at wk 5 and 10 of life, respectively. Feeding did not affect the blood levels of aspartate aminotransferase, gamma glutamyl transferase, glutamate dehydrogenase, and cortisol. In conclusion, feeding high-quality hay, instead of concentrate-rich starter feeds, resulted in improved rumination and ruminal fermentation profile, without affecting ruminal pH and systemic and stress health variables.

Replacing concentrates with a high-quality hay in the starter feed in dairy calves: I. Effects on nutrient intake, growth performance, and blood metabolic profile.

Concentrate-rich starter feeds are commonly fed to dairy calves to stimulate early solid feed intake and growth performance; yet, starter feeds lacking in forage fiber may jeopardize gut development. This research primarily aimed to test a complete or partial replacement of concentrates with hay of different qualities in the starter feed on nutrient intake, growth performance, apparent total-tract digestibility (ATTD) of nutrients, and blood metabolites in dairy calves. Immediately after birth, 40 Holstein Friesian calves were randomly allocated to 1 of 4 starter diets, which differed in hay quality and concentrate inclusion [MQH = 100% medium-quality hay, 9.4 MJ of metabolizable energy (ME), 149 g of crude protein (CP), 522 g of neutral detergent fiber (NDF)/kg of dry matter (DM); HQH = 100% high-quality hay, 11.2 MJ of ME, 210 g of CP, 455 g of NDF/kg of DM; MQH+C = 30% medium-quality hay + 70% starter concentrate; HQH+C = 30% high-quality hay + 70% starter concentrate]. The concentrate consisted mainly of grains, oilseeds, and mineral supplements (13.5 MJ of ME, 193 g of CP, 204 g of NDF/kg of DM). Calves were used in the experiment from d 1 to 99 of life. During the first 4 wk, all calves were fed acidified whole milk ad libitum, and afterward they were gradually weaned from wk 5 to 12. Calves had ad libitum access to their starter diets and water throughout the experiment. Milk, water, and solid feed intake was recorded daily, live weight was measured once a week, and blood samples were collected on d 1, 3, 7, 21, 49, 77, and 91 and analyzed for selected metabolites. The ATTD was measured in wk 14 of life. Total DM intake and daily weight gain of calves were not affected by the starter feed during the first 8 wk of life. However, from wk 9 to 14, calves fed the MQH diet had lower DM, ME, and CP intake and gained less weight than calves from the other experimental groups. Feeding the HQH diet resulted in similar CP and ME intake and growth performance compared with calves receiving diets containing concentrates. Furthermore, feeding the HQH diet improved the ATTD of NDF, resulting in similar ATTD of organic matter with the HQH+C and MQH+C groups. Interestingly, calves fed the HQH+C diet showed less sorting for concentrate, compared with the MQH+C group. Concentration of blood metabolites, including glucose, lactate, insulin, nonesterified fatty acids, triglycerides, and total protein, did not differ after the first week of life. However, serum ß-hydroxybutyrate was higher in calves fed the HQH diet starting from wk 11. Both groups fed the hay-only diets maintained higher cholesterol levels after weaning compared with the groups fed hay-concentrate mixtures. In conclusion, feeding high-quality hay can fully replace starter concentrates in the feeding of dairy calves without adverse effects on performance during the rearing period, while increasing forage fiber intake and utilization, which enhanced ruminal ketogenesis and cholesterogenesis around weaning. Further research is needed to evaluate long-term effects of feeding high-quality hay on health and development of dairy calves, especially in terms of the observed improvements in ruminal ketogenesis and cholesterogenesis around weaning.

Feeding dairy cows bakery by-products enhanced nutrient digestibility, but affected fecal microbial composition and pH in a dose-dependent manner.

We reported recently that adding bakery by-products (BP) to the diets of dairy cows up to 30% improved performance and rumen pH, but caused major shifts in the nutrient profile and availability, likely modifying nutrient degradation patterns throughout the gastrointestinal tract. The aim of this study was to investigate the effects of the gradual replacement of cereals by BP on the apparent total-tract digestibility (ATTD), the fermentation patterns, and the microbial community in feces of dairy cows. Twenty-four mid-lactating Simmental cows (149 ± 22.3 days in milk, 756 ± 89.6 kg of initial body weight) were fed a total mixed ration ad libitum (fresh feed was offered twice per day) containing a 50:50 ratio of forage to concentrate (dry matter basis) throughout the experiment. The trial lasted 5 wk, whereby the first week was used for baseline measurements, in which all cows received the same diet, without BP. Cows were then randomly allocated into 3 groups differing in the BP content of diets (0% BP, 15% BP, and 30% BP on a DM basis) and fed for 4 wk. Fecal samples were taken for analysis of pH, volatile fatty acids (VFA), and 16S rRNA gene sequencing. The inclusion of BP resulted in an increase of ether extract and sugars, and a reduction of starch and neutral detergent fiber in the diet. Feeding BP linearly increased the ATTD of almost all nutrients resulting in up to 2 kg more digestible organic matter intake (DOMI). Increasing BP level up to 30% increased fecal total VFA concentration and decreased the pH. The proportion of butyrate in feces increased linearly, but the proportion of all other VFA was not affected by BP-feeding. The richness and diversity indices of the fecal microbiota linearly declined by the inclusion of BP. The cellulolytic phyla Fibrobacteres decreased, whereas amylolytic phyla, such as Proteobacteria, increased. Overall, results showed that feeding BP linearly increased ATTD and DOMI, but impaired fecal microbial diversity and pH. In the interest of the optimization of BP inclusion in the dairy cows' feeding, a dietary level between 15 to 30% of BP might be a better compromise than 30% in terms of an enhanced DOMI and performance with still lowered risk of hindgut dysbiosis, but this will require further investigations.

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.

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.

Distinct serum metabolomic signatures of multiparous and primiparous dairy cows switched from a moderate to high-grain diet during early lactation.

INTRODUCTION: Feeding of high-grain diets is common in cows during early lactation, but increases the odds of metabolic derailments, which can likely be detected as undesirable shifts in the serum metabolome signature. OBJECTIVES: The present study aimed to identify the metabolic signatures of the serum metabolome of early lactation dairy cows switched from a moderate to a high-grain diet. METHODS: Targeted ESI-LC-MS/MS-based metabolomics was used to characterize metabolic alterations in the serum of early lactation multiparous (MP, n = 16) and primiparous (PP, n = 8) Simmental cows, according to parity and feeding phase. Data were analysed using different data mining approaches. RESULTS: Carnitine, acetylcarnitine, propionoylcarnitine, amino acid related compounds cis-4-hydroxyproline, trans-4-hydroxyproline, proline betaine, lysophosphatidylcholine PC a C16:1 and phosphatidylcholine PC ae C36:0 were identified as the key metabolites distinguishing MP from PP cows. A different serum metabolite composition during moderate and high-grain diet was also evident. Notably, cows fed high grain diet had higher serum concentrations of primary bile acids and triglycerides, but lower levels of conjugated bile acids and carboxylic acids during the first week in grain. Amino acids valine, cystine and taurine together with lysophosphatidylcholine PC a C26:0 and several phosphatidylcholines were classified as important features for cluster separation. CONCLUSIONS: Our study greatly expands earlier observations on dietary effects on serum metabolome composition of cows. The altered metabolomic fingerprints clearly distinguishable by diet and cow parity hold potential to be used as early diagnostic tools for cows experiencing grain-induced metabolic disturbances.

Feeding of bakery by-products in the replacement of grains enhanced milk performance, modulated blood metabolic profile, and lowered the risk of rumen acidosis in dairy cows.

Leftover bakery by-products (BP) from bakeries and supermarkets may serve as energy-rich ingredient in ruminant diets. The aim of the present study was to evaluate the effect of the successive substitution of cereal grains by BP on dry matter (DM) intake, milk production, and metabolic health as well as ruminal pH and eating and chewing behavior of dairy cows. Twenty-four lactating Simmental cows (149 ± 22.3 d in milk, lactation number 2.63 ± 1.38, 756 ± 89.6 kg of initial body weight) were fed a total mixed ration containing a 50:50 ratio of forage to concentrate throughout the experiment (35 d). During the first week, all cows received a control diet (without BP) as a baseline (d -7 to 0). In the next 4 wk (d 1 to 28), cows were allocated to 3 groups differing in the BP concentrations of diets [0% BP (CON), 15% BP, and 30% BP on a DM basis]. The DM intake and reticuloruminal pH were continuously measured. Blood and milk samples were taken every week, but only results from the experimental period (d 21 and 28) were used for statistical analyses, whereas results from the baseline were considered covariates. Diet analyses showed that BP inclusion increased the ether extract and sugar contents, whereby starch and neutral detergent fiber decreased. Experimental data showed that feeding BP in the diet increased DM intake. Furthermore, the cows fed 30% BP produced roughly 4 kg/d more milk and energy-corrected milk than the CON cows. The milk urea nitrogen was lower in cows fed the BP. Feeding BP reduced the blood glucose and insulin concentrations, whereas nonesterified fatty acids, ß-hydroxybutyrate, and cholesterol increased linearly. Cows fed 15% BP had the shortest period of time in which ruminal pH was below 5.8, in contrast to CON cows (+188 min/d). Taken together, the results suggest that the inclusion of up to 30% BP in the diets of mid-lactation dairy cows shifted the nutrient profile from a glucogenic diet to a lipogenic diet, holding the potential to enhance performance and lower the risk of subacute ruminal acidosis in dairy cows.

Distinct responses in feed sorting, chewing behavior, and ruminal acidosis risk between primiparous and multiparous Simmental cows fed diets differing in forage and starch levels.

During early lactation, both primiparous (PP) and multiparous (MP) cows are commonly fed diets rich in starch and low in forages to support their high energy requirements. Yet, the PP cows experience this dietary challenge for the first time, which might result in higher odds for them to develop rumen and systemic health disorders. The primary objective of this study was to evaluate the effect of decreasing the amount of forages in the diet on chewing and sorting behaviors and rumen and systemic health variables in PP and MP dairy cows. Twenty-four lactating Simmental cows [8 PP, average dry matter intake (DMI) of 19.1 ± 1.1 kg/d; 16 MP, average DMI of 22.5 ± 1.1 kg/d] with a body weight of 737 ± 90 kg and 50 ± 22 days in milk were used in this study. Cows were first fed a total mixed ration with 60% forage and 40% concentrate [on a dry matter (DM) basis] considered marginal in forages for 2 wk. Then, cows were switched to a diet low in forages with 40% forage and 60% concentrate (on a DM basis) for 4 wk. Reticular pH was measured continuously with wireless pH-sensors inserted into the reticulum to calculate the subacute ruminal acidosis (SARA) index. Chewing activity was measured with noseband-sensor halters, and feed sorting was measured weekly. Blood samples were collected weekly and analyzed for metabolic and inflammation markers. Switching PP and MP cows from a marginal to low-forage diet decreased the time spent eating and ruminating per kilogram of DM. Primiparous cows chewed longer per kilogram of DMI than MP cows. Also, the PP cows sorted more pronounced for longer particles and against fine particles than MP cows did. Despite higher rumination activity per kilogram of DMI and the adaptive sorting behavior, the PP cows spent on average 4.6 h/d longer below a pH of 5.8 and had a higher SARA index (i.e., area pH <5.8/DMI) than MP cows, especially during the first week of the low-forage diet (9.5 vs. 4.8). The concentration of liver enzymes increased with the low-forage diet, which was especially pronounced in the PP cows. In conclusion, this study demonstrated greater susceptibility of PP cows to SARA and liver damage than MP cows fed the same diets. Although PP cows demonstrated greater chewing and ruminating activity per kilogram of DMI, as well as adapted sorting behavior in favor of large particles during the low-forage high-starch feeding, they developed more severe signs of SARA. This suggests higher forage fiber requirements for PP cows and the need for improved feeding strategies to mitigate rumen fermentation disorders during early lactation in these cows.

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.

Supplementation of a clay mineral-based product modulates plasma metabolomic profile and liver enzymes in cattle fed grain-rich diets.

Grain-rich diets often lead to subacute ruminal acidosis (SARA) impairing rumen and systemic cattle health. Recent data suggest beneficial effects of a clay mineral (CM)- based product on the rumen microbiome of cattle during SARA. This study sought to investigate whether the CM supplementation can counteract SARA-induced perturbations of the bovine systemic health. The study used an intermittent diet-induced SARA-model with eight dry Holstein cows receiving either no additive as control or CM via concentrates (n=8 per treatment). Cows received first a forage diet (Baseline) for 1 week, followed by a 1-week SARA-challenge (SARA 1), a 1-week recovery phase (Recovery) and finally a second SARA-challenge for 2 weeks (SARA 2). Cows were monitored for feed intake, reticular pH and chewing behavior. Blood samples were taken and analyzed for metabolites related to glucose and lipid metabolism as well as liver health biomarkers. In addition, a targeted electrospray ionization-liquid chromatography-MS-based metabolomics approach was carried out on the plasma samples obtained at the end of the Baseline and SARA 1 phase. Data showed that supplementing the cows' diet with CM improved ruminating chews per regurgitated bolus by 16% in SARA 1 (P=0.01) and enhanced the dry matter intake during the Recovery phase (P=0.05). Moreover, the SARA-induced decreases in several amino acids and phosphatidylcholines were less pronounced in cows receiving CM (P≤0.10). The CM-supplemented cows also had lower concentrations of lactate (P=0.03) and biogenic amines such as histamine and spermine (P<0.01) in the blood. In contrast, the concentration of acylcarnitines with key metabolic functions was increased in the blood of treated cows (P≤0.05). In SARA 2, the CM-cows had lower concentrations of the liver enzymes aspartate aminotransferase and γ-glutamyltransferase (P<0.05). In conclusion, the data suggest that supplementation of CM holds the potential to alleviate the negative effects of high-grain feeding in cattle by counteracting multiple SARA-induced perturbations in the systemic metabolism and liver health.

Innate immunity and metabolomic responses in dairy cows challenged intramammarily with lipopolysaccharide after subacute ruminal acidosis.

Subacute ruminal acidosis (SARA) is a prevalent metabolic disorder in dairy cows known to elicit local and systemic immune responses. We recently showed that cows experiencing SARA and challenged intramammarily with lipopolysaccharide (LPS) experienced stronger metabolic disturbances compared with cows without SARA. Therefore, we hypothesized that cows experiencing SARA have a modulated innate immune response and impaired plasma metabolome compared with healthy cows when experiencing an acute mastitis challenge. A total of 18 Simmental cows were subjected either to a Control (CON, n=6) or SARA (n=12) feeding regimen, receiving either 40% or 60% concentrates for 30 days. Thereafter, six SARA (SARA-LPS) and the CON (CON-LPS) cows were intramammarily challenged with 50 µg LPS from Escherichia coli (O26 : B6), while the remaining six SARA cows (SARA-PLA) received a placebo. Blood and milk samples were analyzed for acute phase proteins and a targeted ESI-LC-MS/MS-based metabolomics approach was performed in blood samples 24 h after the LPS challenge. The LPS infusion caused a strong increase in immune response variables, with a higher concentration of milk amyloid A 48 h after the LPS challenge in SARA-LPS compared with CON-LPS cows. Cows receiving the LPS infusion had a lower plasma concentration of several amino acids and lysophosphatidylcholines but without differences in SARA cows and healthy cows. In conclusion, our results revealed that an intramammary LPS infusion increased acute phase proteins and modulated the blood metabolome. While no systemic differences between SARA and healthy cows were observed, cows experiencing SARA showed a higher concentration of an acute phase protein at the local level of the mammary gland. Further research is required to elucidate the underlying mechanisms and to evaluate its clinical significance for udder health.

Review: Enhancing gastrointestinal health in dairy cows.

Due to their high energy requirements, high-yielding dairy cows receive high-grain diets. This commonly jeopardises their gastrointestinal health by causing subacute ruminal acidosis (SARA) and hindgut acidosis. These disorders can disrupt nutrient utilisations, impair the functionalities of gastrointestinal microbiota, and reduce the absorptive and barrier capacities of gastrointestinal epithelia. They can also trigger inflammatory responses. The symptoms of SARA are not only due to a depressed rumen pH. Hence, the diagnosis of this disorder based solely on reticulo-rumen pH values is inaccurate. An accurate diagnosis requires a combination of clinical examinations of cows, including blood, milk, urine and faeces parameters, as well as analyses of herd management and feed quality, including the dietary contents of NDF, starch and physical effective NDF. Grain-induced SARA increases acidity and shifts availabilities of substrates for microorganisms in the reticulo-rumen and hindgut and can result in a dysbiotic microbiota that are characterised by low richness, diversity and functionality. Also, amylolytic microorganisms become more dominant at the expense of proteolytic and fibrolytic ones. Opportunistic microorganisms can take advantage of newly available niches, which, combined with reduced functionalities of epithelia, can contribute to an overall reduction in nutrient utilisation and increasing endotoxins and pathogens in digesta and faeces. The reduced barrier function of epithelia increases translocation of these endotoxins and other immunogenic compounds out of the digestive tract, which may be the cause of inflammations. This needs to be confirmed by determining the toxicity of these compounds. Cows differ in their susceptibility to poor gastrointestinal health, due to variations in genetics, feeding history, diet adaptation, gastrointestinal microbiota, metabolic adaptation, stress and infections. These differences may also offer opportunities for the management of gastrointestinal health. Strategies to prevent SARA include balancing the diet for physical effective fibre, non-fibre carbohydrates and starch, managing the different fractions of non-fibre carbohydrates, and consideration of the type and processing of grain and forage digestibility. Gastrointestinal health disorders due to high grain feeding may be attenuated by a variety of feed supplements and additives, including buffers, antibiotics, probiotics/direct fed microbials and yeast products. However, the efficacy of strategies to prevent these disorders must be improved. This requires a better understanding of the mechanisms through which these strategies affect the functionality of gastrointestinal microbiota and epithelia, and the immunity, inflammation and 'gastrointestinal-health robustness' of cows. More representative models to induce SARA are also needed.

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.

Changes in the Rumen Epithelial Microbiota of Cattle and Host Gene Expression in Response to Alterations in Dietary Carbohydrate Composition.

The inclusion of high-quality hay (HQH), in place of concentrates, shifts dietary carbohydrate intake, and the extent to which these shifts effect epimural microbiota and epithelial gene expression of the rumen has not yet been evaluated. Eight ruminally cannulated nonlactating Holstein cows were used in a replicated 4 by 4 Latin square design with four dietary treatments containing HQH, with either 0% concentrate/100% HQH (100HQH), 25% concentrate/75% HQH (75HQH), or 40% concentrate/60% HQH (60HQH). The fourth group (control [CON]) was fed 60% normal fiber-rich hay and 40% concentrate. The data showed that measures of diversity for the rumen epimural population, specifically the Shannon (P = 0.004) and Simpson (P = 0.003) indices, decreased with increasing levels of HQH in the diet. The feeding of HQH shifted the epimural population from predominantly Firmicutes to Proteobacteria Phylogenetic analysis revealed that HQH feeding markedly shifted the abundance of Campylobacter spp. from 7.8 up to 33.5% (P < 0.001), with greater ingestion of protein (r = 0.63) and sugars (r = 0.65) in HQH diet being responsible for this shift. The expression of genes targeting intracellular pH regulation, barrier function, and nutrient uptake of rumen epithelium remained stable regardless of the carbohydrate source. In conclusion, the data suggest strong alterations of the ruminal epimural microbiota in response to changes in the nutritive patterns of the diet. Further research is warranted to evaluate the long-term effects of these significant microbial changes on rumen health and food safety aspects in cattle at a transcriptional level.IMPORTANCE Feeding of forages versus starchy concentrates is a highly debated topic. Hay is believed to be healthier and more ecological sustainable for cattle than are concentrates, although the effects of feeding hay with enhanced sugar and protein content on epimural microbiota and host gene expression have not yet been evaluated. This research provides a report of the role of feeding hay with increased sugar and protein content in place of starchy concentrates in altering epimural microbiota and in generating a host response. Our research shows that the addition of high-quality hay to dairy rations shifted nutrient intake, resulting in strong alterations in the epimural microbiota in cattle. This work provides a background for further long-term research regarding the effects of feeding practices on the host-microbiome interaction and its role in rumen health and food safety in cattle.

Graded substitution of grains with bakery by-products modulates ruminal fermentation, nutrient degradation, and microbial community composition in vitro.

A new segment of feed industry based on bakery by-products (BBP) has emerged. Yet, information is lacking regarding the effects of inclusion of BBP in ruminant diets on ruminal fermentation and microbiota. Therefore, the aim of this study was to evaluate the effect of the gradual replacement of grains by BBP on ruminal fermentation, nutrient degradation, and microbial community composition using the rumen-simulation technique. All diets consisted of hay and concentrate mixture with a ratio of 42:58 (dry matter basis), but differed in the concentrate composition with either 45% cereal grains or BBP, whereby 15, 30, or 45% of BBP were used in place of cereal grains. The inclusion of increasing levels of BBP in the diet linearly enhanced ruminal degradation of starch from 84% (control) to 96% (45% BBP), while decreasing degradation of crude protein and fiber. The formation of methane was lowered in the 45% BBP diet compared with all other diets. Whereas the ammonia concentration was similar in the control and 15% BBP, a significant decrease was found in 30% BBP (-23%) and 45% BBP (-33%). Also, BBP feeding shifted fermentation profile toward propionate at the expense of acetate. Moreover, isobutyrate linearly decreased with increasing BBP inclusion. Bacterial 16S rRNA Illumina MiSeq (Microsynth AG, Balach, Switzerland) sequencing revealed a decreased microbial diversity for the 45% BBP diet. Furthermore, the replacement of cereal grains with BBP went along with an increased abundance of the genera Prevotella, Roseburia, and Megasphaera, while decreasing Butyrivibrio and several OTU belonging to Ruminococcaceae. In conclusion, the inclusion of BBP at up to 30% of the dry matter had no detrimental effects on pH, fiber degradability, and microbial diversity, and enhanced propionate production. However, a higher replacement level (45%) impaired ruminal fermentation traits and fiber degradation and is not recommended.

High-grain diets supplemented with phytogenic compounds or autolyzed yeast modulate ruminal bacterial community and fermentation in dry cows.

The feeding of concentrate-rich diets may lead to microbial imbalances and dysfermentation in the rumen. The main objective of this study was to determine the effects of supplementing phytogenic compounds (PHY) or autolyzed yeast (AY) on rumen fermentation and microbial abundance in cows intermittently fed concentrate-rich diets. The experiment was carried out as an incomplete 3 × 4 Latin square design, with 8 nonlactating rumen-fistulated Holstein-Friesian cows. The cows were randomly assigned to a concentrate diet that was either not supplemented (CON), or supplemented with PHY or AY. Each of the 4 consecutive experimental periods was composed of a 1-wk roughage-only diet (RD), 6-d gradual concentrate increase, followed by 1 wk of 65% concentrate (dry matter basis; Conc I), and 1 wk of RD and a final 2-wk 65% concentrate (dry matter basis; Conc II) phase. Digesta samples were collected from the rumen mat for bacterial 16S rRNA gene Illumina MiSeq (Illumina, Balgach, Switzerland) sequencing, and samples of particle-associated rumen liquid were obtained for measuring short-chain fatty acids, lactate, ammonia, and pH during RD (d 6), Conc I (d 19), and Conc II (d 39). The concentrate feeding caused a decrease of overall bacterial diversity indices, especially during Conc I. The genera Ruminococcus, Butyrivibrio, and Coprococcus were decreased, whereas Prevotella, Megasphaera, Lachnospira, and Bacteroides were increased in abundance. Supplementation of both feed additives increased the abundance of gram-positive and decreased that of gram-negative bacteria. Supplementation of AY enhanced cellulolytic bacteria such as Ruminococcus spp., whereas PHY decreased starch and sugar fermenters including Bacteroides spp., Shuttleworthia spp., and Syntrophococcus spp. Moreover, PHY supplementation increased butyrate percentage in the rumen in both concentrate phases. In conclusion, intermittent high-concentrate feeding altered the digesta-associated rumen bacterial community and rumen fermentation with more significant alterations found in Conc I than in Conc II. The data also showed that both feed additives had the most significant modulatory effects on the bacterial community, and their subsequent fermentation, during periods of low pH.

Differences between pH of indwelling sensors and the pH of fluid and solid phase in the rumen of dairy cows fed varying concentrate levels.

Feeding of high-concentrate diets to cattle increases the risk of subacute ruminal acidosis (SARA). Indwelling wireless pH sensors have become popular as an early diagnostic tool for SARA recently. However, the recommended pH thresholds of SARA derive from measurements taken from free-rumen liquid (FRL) in the ventral rumen, and not from the reticulum, where the mentioned sensors are located. The aim of this study was to evaluate commercially available indwelling pH boli for the accuracy and precision in predicting ruminal pH of FRL and the particle-associated rumen liquid (PARL) under varying dietary concentrate levels. An additional aim was to define SARA pH thresholds of indwelled pH boli, which can be used for SARA diagnostics. The experiment was conducted with eight nonlactating rumen cannulated Holstein cows fed 0% or 65% concentrate. Data showed that the mean pH of indwelled boli was consistently higher than in FRL and PARL (pH 6.62 ± 0.02, 6.43 ± 0.02 and 6.18 ±0.02, respectively) across feeding regimens. Interestingly, the diurnal differences in pH among indwelled boli, FRL and PARL became greater during concentrate feeding, especially at 8 h after the morning feeding, suggesting that with high-concentrate diets a particular adjustment of reticular sensor pH vs. ruminal pH in FRL and PARL is needed. The concordance correlation coefficient analysis, representing the reproducibility of the bolus measurements, was high for bolus-FRL (0.733) and moderate for bolus-PARL (0.510) associations. Furthermore, the quantitative relationship of the pH in FRL and PARL to the pH of the boli was described by linear regression analysis. The study determined that the common SARA threshold of pH 5.8 in FRL corresponds to a bolus pH of 6.0.

Metabolic and stress responses in dairy cows fed a concentrate-rich diet and submitted to intramammary lipopolysaccharide challenge.

Feeding dairy cows diets rich in grain often leads to subacute rumen acidosis (SARA), which might affect their responsiveness to immunogenic stimuli such as exogenous lipopolysaccharide (LPS), and can lead to metabolic alterations. The main objective of this study was to investigate if SARA affects the stress and metabolic health responses resulting from an intramammary LPS challenge. Before the intramammary LPS challenge, the SARA cows showed higher blood glucose and a tendency for higher lactate and aspartate aminotransferase as well as a trend toward lower ß-hydroxybutyrate (BHBA) and γ-glutamyltransferase compared with control cows. After the LPS challenge, the serum cortisol concentration markedly increased and the calcium concentration decreased both in SARA and control cows. In SARA-LPS cows, however, the lactate concentration increased due to the LPS infusion, whereas it remained unchanged in the control cows. A lower serum BHBA concentration was found in SARA-LPS compared with control-LPS cows. Higher non-esterified fatty acid concentrations were found in control-LPS cows shortly before the LPS challenge compared with SARA cows, challenged or not with LPS, whereas it did not differ from SARA-LPS cows thereafter. In conclusion, the results suggest that intramammary LPS challenge induced stress and lowered calcium concentration in all dairy cows, whereby this challenge showed lower BHBA and higher lactate levels in cows with SARA conditions.