Prolonged feeding of high-concentrate diet remodels the hindgut microbiome and modulates nutrient degradation in the rumen and the total gastrointestinal tract of cows.

The aims of this research were to evaluate how prolonged feeding of a high-concentrate diet affects the ruminal degradation kinetics of fiber and starch, and to evaluate the effects of the high-concentrate diet on apparent total-tract nutrient digestibility in dairy cows. We also investigated the dysbiotic effects and the remodeling of the hindgut microbiome with prolonged high-concentrate feeding. Nine Holstein cows were used in 2 experimental periods; in each period, cows were first fed a 100% forage diet (Forage) for 1 week, followed by stepwise adaptation during one week to a high-concentrate diet (HC; 65% concentrate), which was then fed for 4 consecutive weeks. The kinetics of in situ ruminal degradability of grass silage (DM and NDF), corn grain and wheat grain (DM and starch) as well as the apparent total-tract nutrient digestibility were evaluated in the Forage feeding and in wk 4 on HC. Whereas the hindgut microbiome and fermentation profile were evaluated on a weekly basis. Regarding the in situ ruminal degradability due to grain type, the rate of degradation of the potentially degradable fraction of the grain and the effective rumen degradability of wheat grain were greater compared with corn grain. The in situ ruminal degradability of NDF decreased with the HC diet. However, the apparent total-tract digestibility of crude protein, fat, starch, NDF, ADF and NFC increased with HC compared with Forage feeding. In addition, the HC diet increased the concentration of short-chain fatty acids in the hindgut, lowering fecal pH by 0.6 units, which correlated positively with microbial α diversity. This resulted in lower α diversity with HC; however, α diversity (number of ASVs) showed recovery in wk 3 and 4 on HC; in addition, microbial ß diversity did not change from wk 2 on HC onwards. Two microbial enterotypes were identified: one for the Forage diet with abundance of Akkermansia and Anaerosporobacter, and another enterotype for the HC diet with enrichment in Bifidobacterium and Butyrivibrio. Overall, results show that major microbial shifts and hindgut dysbiosis occurred in wk 1 on HC. However, the hindgut microbial diversity of cows adapted after 3 weeks of consuming the starch-rich ration. Thus, feeding HC diet impaired fiber degradation in the rumen, but increased apparent total-tract nutrient digestibility. Likely, the forage diet contained less digestible NDF than the HC diet due to greater inclusion of forages with lower NDF digestibility and lower inclusion of more digestible non-forage NDF. Results also suggest that the adaptation of the hindgut microbial diversity of cows observed 3 weeks after the diet transition likely contributed to enhance total-tract nutrient digestibility.

Duration of Zearalenone Exposure Has Implications on Health Parameters of Lactating Cows.

There is a limited research focus on evaluating the detrimental effects of prolonged zearalenone (ZEN) intake on dairy cows' health under controlled conditions. This experiment was conducted to evaluate whether the length of exposure to a ZEN-contaminated total mixed ration (TMR) at a level of 9.45 mg per day can negatively influence animal health parameters, such as milk composition, rumen and fecal fermentation, and the chewing activity of lactating dairy cows. For this experiment, we used 18 lactating Simmental cows that were fed a diet of 60% forage and 40% concentrate (on dry matter basis) for 26 consecutive days. The first 4 days were for adaptation prior to the first sampling day (day 0). The sampling events took place on day 0 (baseline) without ZEN, followed by day 1, day 7, day 14, and day 21 (with toxin). Dry matter intake (DMI) and ruminating chews per minute increased on the third week of ZEN inclusion; meanwhile, ruminating, eating, and drinking times were not affected. Most milk composition variables were also unaffected. Rumen fluid osmolality increased on day 21 and total short-chain fatty acids (SCFA) of ruminal fluid decreased on day 7. Fecal SCFA increased on day 21 and the acetate-to-propionate ratio increased from day 1 onwards, showing the influence of toxin intake. Animal health parameters, like heart rate, respiratory rate, and body temperature, were negatively influenced by ZEN intake, all increasing consistently on days 4 and 6, 9 and 12, and 16 and 18, respectively. The liver enzyme glutamate dehydrogenase decreased in response to ZEN intake on day 7. A total daily ZEN intake at the level of 9.45 mg did not show detrimental effects on DMI. Nevertheless, certain health parameters were negatively affected, including body temperature, respiratory rate, and heart rate, starting from the 7th day of ZEN intake, with additional signs of possible loss of water balance on the last sampling day.

Correction: Penagos-Tabares et al. Mixtures of Mycotoxins, Phytoestrogens, and Other Secondary Metabolites in Whole-Plant Corn Silages and Total Mixed Rations of Dairy Farms in Central and Northern Mexico. Toxins 2023, 15, 153.

In the original publication [...].

Elucidating the factors and consequences of the severity of rumen acidosis in first-lactation Holstein cows during transition and early lactation.

First-lactation cows are particularly prone to subacute ruminal acidosis (SARA) during transition. Besides common risk factors of SARA, such as feeding of starch-rich diets, an individual severity of SARA in cows has been recently evidenced. Yet, the factors that play a role in SARA severity have not been elucidated. The main goal of this research was to evaluate the factors of SARA severity in first-lactation cows during transition and early lactation, which go beyond high-grain feeding, and to explore their impact on behavior, health, and fermentation in the rumen and hindgut. Twenty-four first-lactation Holstein cows with the same feeding regime were used starting from 3 wk before the expected calving day until 10 wk postpartum. Cows received a close-up diet (32% concentrate) until calving and were then transitioned to a lactation diet (60% concentrate) within 1 week. The SARA severity was assessed by cluster analysis of several rumen pH metrics, which revealed exceptionally longer and more severe SARA in cows denominated as high (n = 9), as compared to moderate (n = 9) and low (n = 6) SARA severity cows (P < 0.01). The logistic analysis showed that the length of close-up feeding, age at parturition, and the level of dry matter intake (DMI) were the main factors that influenced the cows' odds for high SARA severity (each P ≤ 0.01). Moreover, the ANOVA hinted differences in the metabolic activity of the ruminal microbiome to promote SARA severity, as indicated by highest ruminal propionate proportions (P = 0.05) in high SARA severity cows, also with similar DMI. The distinct SARA severity was marginally reflected in behavior and there were no effects of SARA severity or high-grain feeding on blood inflammation markers, which peaked at parturition regardless of SARA severity (P < 0.01). Still, ongoing high-grain feeding increased liver enzyme concentrations from 6 wk postpartum on, compared to weeks before (P < 0.01), yet irrespectively of SARA severity. In conclusion, first-lactation cows differed in SARA severity under the same feeding regime, which was ascribed to management factors and differences in ruminal fermentation. Further research is warranted to validate these findings and to understand the mechanisms behind differences in the metabolic function of rumen microbiome, in particular in terms of evaluating markers for various SARA severity, as well as to evaluate potential long-term effects on health, performance, fertility, and longevity of dairy cows.

The present study reports a high variation of severity of subacute rumen acidosis in first-lactation dairy cows with the same feeding regimen close to parturition and until 10 weeks after parturition. Six significant factors influencing this severity were identified, including in particular length of close-up period, age at parturition, and dry matter intake. Therefore, management factors seem to play a key role for the development of a severe subacute rumen acidosis. Cows with high severity showed marginally altered behavior but distinct rumen fermentation patterns compared to cows with low severity, suggesting also a key role of the ruminal microbiome for subacute rumen acidosis risk in cows. The higher severity was not associated with systemic inflammation and all cows remained healthy.

Comparison of LC-MS-based methods for the determination of carboxylic acids in animal matrices.

Carboxylic acids (CAs) are key players in human and animal metabolism. As they are hardly retained under reversed-phase liquid chromatography (RP-LC) conditions in their native form, derivatization is an option to make them accessible to RP-LC and simultaneously increase their response for mass spectrometric detection. In this work, two RP-LC tandem mass spectrometry-based methods using aniline or 3-nitrophenylhydrazine (3-NPH) as derivatization agents were compared with respect to several factors including completeness of derivatization, apparent recoveries (RAs) in both cow feces and ruminal fluid, and concentrations obtained in feces and ruminal fluid of cows. Anion exchange chromatography coupled to high-resolution mass spectrometry (AIC-HR-MS) served as reference method. Derivatization efficiencies were close to 100% for 3-NPH derivatization but variable (20-100%) and different in solvent solutions and matrix extracts for aniline derivatization. Likewise, average RAs of 13C-labeled short-chain fatty acids as internal standards were around 100% for 3-NPH derivatization but only 45% for aniline derivatization. Quantification of CAs in feces and ruminal fluid of cows initially fed a forage-only diet and then transitioned to a 65% high-grain diet which yielded similar concentrations for 3-NPH derivatization and AIC-HR-MS, but concentrations determined by aniline derivatization were on average five times lower. For these reasons, derivatization with aniline is not recommended for the quantitative analysis of CAs in animal samples.

Integrated microbiota-host-metabolome approaches reveal adaptive ruminal changes to prolonged high-grain feeding and phytogenic supplementation in cattle.

Diets rich in readily fermentable carbohydrates primarily impact microbial composition and activity, but can also impair the ruminal epithelium barrier function. By combining microbiota, metabolome, and gene expression analysis, we evaluated the impact of feeding a 65% concentrate diet for 4 weeks, with or without a phytogenic feed additive (PFA), on the rumen ecosystem of cattle. The breaking point for rumen health seemed to be the second week of high grain (HG) diet, with a dysbiosis characterized by reduced alpha diversity. While we did not find changes in histological evaluations, genes related with epithelial proliferation (IGF-1, IGF-1R, EGFR, and TBP) and ZO-1 were affected by the HG feeding. Integrative analyses allowed us to define the main drivers of difference for the rumen ecosystem in response to a HG diet, identified as ZO-1, MyD88, and genus Prevotella 1. PFA supplementation reduced the concentration of potentially harmful compounds in the rumen (e.g. dopamine and 5-aminovaleric acid) and increased the tolerance of the epithelium toward the microbiota by altering the expression of TLR-2, IL-6, and IL-10. The particle-associated rumen liquid microbiota showed a quicker adaptation potential to prolonged HG feeding compared to the other microenvironments investigated, especially by the end of the experiment.

Influence of Azadirachta indica and Cnidoscolus angustidens aqueous extract on cattle ruminal gas production and degradability in vitro.

Introduction: Mitigation of ruminant greenhouse gas (GHG) emissions is crucial for more appropriate livestock production. Thus, there is a need of further research evaluating feed supplementation strategies to mitigate enteric GHG emissions and other gases produced within the rumen. Methods: This study was conducted as a completely randomized experimental design to determine the effectiveness of liquid extracts from A. indica (AZI), C. angustidens (CNA), or their combination (Mix. 1:1) at dosages of 0, 36, 72, and 108 mg of liquid extract/g DM substrate incubated in reducing GHG production in vitro, particularly methane (CH4), from the diet of steers during anaerobic incubation in rumen fluid. Total gas production, CH4, CO, H2S, and fermentative characteristics were all measured in vitro. Results: Treatment AZI at a dose of 108 mg of liquid extract/g DM substrate produced the highest (P < 0.05) gas volume at 6 h, whereas CNA at a dose of 72 mg of liquid extract/ g DM substrate produced the least (P < 0.05) at 6 and 24 h, and Mix. at a dose of 72 mg of liquid extract/g DM substrate produced the least (P < 0.05) at 48 h. In addition, CH4 levels at 6 and 24 h of incubation (36 mg/g DM substrate) were highest (P < 0.05) for CNA, and lowest (P < 0.05) for AZI, whereas this variable was lowest (P < 0.05) at 72 mg of liquid extract for CNA at 24 and 48 h. At 6 and 24 h, CO volume was highest (P < 0.05) for AZI at 108 mg of liquid extract and lowest (P < 0.05) for Mix. at 72 mg of liquid extract. Treatment Mix. had a high (P < 0.05) concentration of short chain fatty acids at 72 mg of liquid extract/g DM of substrate. Discussion: In general, herbaceous perennial plants, such as AZI and CNA, could be considered suitable for mitigating enteric GHG emissions from animals. Specifically, the treatment Mix. achieved a greater sustainable reduction of 67.6% in CH4 and 47.5% in H2S production when compared to either AZI. This reduction in CH4 might suggest the potential of the combination of both plant extracts for mitigating the production of GHG from ruminants.

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

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

Mixtures of Mycotoxins, Phytoestrogens, and Other Secondary Metabolites in Whole-Plant Corn Silages and Total Mixed Rations of Dairy Farms in Central and Northern Mexico.

Mycotoxins and endocrine disruptors such as phytoestrogens can affect cattle health, reproduction, and productivity. Most studies of mycotoxins in dairy feeds in Mexico and worldwide have been focused on a few (regulated) mycotoxins. In contrast, less known fungal toxins, phytoestrogens, and other metabolites have been neglected and underestimated. This study analyzed a broad spectrum (>800) of mycotoxins, phytoestrogens, and fungal, plant, and unspecific secondary metabolites in whole-plant corn silages (WPCSs) and total mixed rations (TMRs) collected from 19 Mexican dairy farms. A validated multi-metabolite liquid chromatography/electrospray ionization-tandem mass spectrometric (LC/ESI-MS/MS) method was used. Our results revealed 125 of >800 tested (potentially toxic) secondary metabolites. WPCSs/TMRs in Mexico presented ubiquitous contamination with mycotoxins, phytoestrogens, and other metabolites. The average number of mycotoxins per TMR was 24, ranging from 9 to 31. Fusarium-derived secondary metabolites showed the highest frequencies, concentrations, and diversity among the detected fungal compounds. The most frequently detected mycotoxins in TMRs were zearalenone (ZEN) (100%), fumonisin B1 (FB1) (84%), and deoxynivalenol (84%). Aflatoxin B1 (AFB1) and ochratoxin A (OTA), previously reported in Mexico, were not detected. All TMR samples tested positive for phytoestrogens. Among the investigated dietary ingredients, corn stover, sorghum silage, and concentrate proportions were the most correlated with levels of total mycotoxins, fumonisins (Fs), and ergot alkaloids, respectively.

Selective decline of bacteria colonizing the calf hindgut during colostrum to milk transition, with persistence of taxa correlating with host passive immunity.

In neonate calves, the association between gut microbial colonization and passive immunity acquisition remains largely unknown. We evaluated the effect of transition from colostrum to milk on the hindgut microbiota, and the correlations between acquired passive immunity and this microbiome. In 14 Holstein calves, colostrum quality and host passive immunity were measured, feces were sampled when feeding colostrum and after transition to milk. Then, in eight calves displaying a wide range of passive immunity, the hindgut microbiota was evaluated with DNA sequencing; differential abundance was analyzed with Maaslin2. With transition from colostrum to milk, many initial bacterial colonizers did not survive; genus Ralstonia decreased, but Lactobacillus and Bacteroides increased. When feeding colostrum, the amount of immunoglobulins consumed positively correlated with abundance of Lactobacillaceae and Lachnospiraceae, but Escherichia-Shigella and Clostridium sensu stricto 1 correlated negatively with host passive immunity. After transition to milk, acquired passive immunity negatively correlated with Clostridium sensu stricto 1, Ralstonia, and Veillonella. Overall, many initial hindgut colonizers did not thrive during transition from colostrum to milk, homogenizing the bacterial profile with prevalence of milk digesters. Several bacterial taxa showed strong correlation with host passive immunity, suggesting an interplay between calf passive immunity acquisition and the colonizing microbiota.

Diet and phytogenic supplementation substantially modulate the salivary proteome in dairy cows.

Phytogenic compounds may influence salivation or salivary properties. However, their effects on the bovine salivary proteome have not been evaluated. We investigated changes in the bovine salivary proteome due to transition from forage to high-concentrate diet, with and without supplementation with a phytogenic feed additive. Eight non-lactating cows were fed forage, then transitioned to a 65% concentrate diet (DM basis) over a week. Cows were control (n = 4, CON) or supplemented with a phytogenic feed additive (n = 4, PHY). Proteomic analysis was conducted using liquid chromatography coupled with mass spectrometry. We identified 1233 proteins; 878 were bovine proteins, 189 corresponded to bacteria, and 166 were plant proteins. Between forage and high-concentrate, 139 proteins were differentially abundant (P < 0.05), with 48 proteins having a log2FC difference > |2|. The salivary proteome reflected shifts in processes involving nutrient utilization, body tissue accretion, and immune response. Between PHY and CON, 195 proteins were differently abundant (P < 0.05), with 37 having a log2FC difference > |2|; 86 proteins were increased by PHY, including proteins involved in smell recognition. Many differentially abundant proteins correlated (r > |0.70|) with salivary bicarbonate, total mucins or pH. Results provide novel insights into the bovine salivary proteome using a non-invasive approach, and the association of specific proteins with major salivary properties influencing rumen homeostasis. SIGNIFICANCE: Phytogenic compounds may stimulate salivation due to their olfactory properties, but their effects on the salivary proteome have not been investigated. We investigated the effect of high-concentrate diets and supplementation with a phytogenic additive on the salivary proteome of cows. We show that analysis of cows' saliva can be a non-invasive approach to detect effects occurring not only in the gut, but also systemically including indications for gut health and immune response. Thus, results provide unique insights into the bovine salivary proteome, and will have a crucial contribution to further understand animal response in terms of nutrient utilization and immune activity due to the change from forage to a high-energy diet. Additionally, our findings reveal changes due to supplementation with a phytogenic feed additive with regard to health and olfactory stimulation. Furthermore, findings suggest an association between salivary proteins and other components like bicarbonate content.

Variations in fecal pH and fecal particle size due to changes in dietary starch: Their potential as an on-farm tool for assessing the risk of ruminal acidosis in dairy cattle.

This study evaluated the variations in fecal pH and particle size due to changes in dietary starch, and the potential of these variations as a tool to detect the risk of subacute ruminal acidosis (SARA) in dairy cows. Nine ruminally cannulated, non-lactating, non-pregnant Holstein cows were used in two 6-week experimental periods. In each period, cows were first fed a forage diet for 1 wk., then transitioned over 1 wk. to a 65% concentrate ration, which they consumed for 4 wk. continuously. Measurements were conducted when cows consumed 17.3, 21.9 and 28.8% dietary starch. Fecal pH and particle size were measured at 0, 4, 8 and 12 h relative to feeding in days 7, 11, 21, 28, 35, and 42 of each period. Ruminal pH was measured every 15 min. Data were analyzed with SAS, the statistical model included concentrate level, time of sampling and period as fixed effects and cow was considered as random effect. Data showed an interaction between dietary starch level and time relative to feeding on fecal pH, with a shift in its pattern due to diet change. Specifically, during low starch feeding (17.3%), fecal pH was highest before feeding and decreased thereafter, reaching lowest value 12 h post-feeding (P < 0.05). With the 21.9% starch diet, fecal pH did not change significantly after feeding. However, during high starch feeding (28.8%), fecal pH decreased, being lowest before feeding and increased (P < 0.05) during the day reaching highest values at 8 and 12 h post-feeding. Fecal pH was lower (P < 0.01) during the days of high starch feeding; though, it was maintained relatively stable through this timeframe. Increasing dietary starch decreased the proportion of small fecal particles (0.5 to 1.18 mm), but increased the proportion of large (>1.18 mm) and soluble particles (<0.5 mm). There were significant correlations among ruminal pH, fecal pH and fecal particle size of feces collected before feeding. For example, fecal pH was correlated with minimum and daily mean ruminal pH; the proportion of fecal particles 0.5 to 1.18 mm correlated with minimum and daily mean ruminal pH (P < 0.01). Log-linear dependency analyses indicated a strong effect of starch intake on fecal pH so that for every kg increase in starch intake, there was a reduction in fecal pH by 0.38 units. In addition, regression analysis showed that the proportion of fecal particles between 0.5 and 1.18 mm showed strong dependency on the ratio dietary physically effective fiber to starch (P < 0.01). Ruminal pH also correlated with fecal particle size of frozen/thawed samples, but with lower strength than fresh samples. Overall, evaluating variations of fecal pH and particle size holds potential as a non-invasive on-farm tool for assessing rumen pH and SARA risk.

Progressive microbial adaptation of the bovine rumen and hindgut in response to a step-wise increase in dietary starch and the influence of phytogenic supplementation.

Microbial composition and activity in the gastrointestinal tract (GIT) of cattle has important implications for animal health and welfare, driving the focus of research toward ways to modify their function and abundance. However, our understanding of microbial adaption to nutritional changes remains limited. The aim of this study was to examine the progressive mechanisms of adaptation in the rumen and hindgut of cattle receiving increasing amounts of starch with or without dietary supplementation of a blended phytogenic feed additive (PFA; containing menthol, thymol and eugenol). We used 16S rRNA gene amplicon sequencing to assess the microbial composition and predicted metabolic pathways in ruminal solid and liquid digesta, and feces. Furthermore, we employed targeted liquid chromatography-mass spectrometry methods to evaluate rumen fluid metabolites. Results indicated a rapid microbial adaptation to diet change, starting on the second day of starch feeding for the particle associated rumen liquid (PARL) microbes. Solid rumen digesta- and feces-associated microbes started changing from the following day. The PARL niche was the most responsive to dietary changes, with the highest number of taxa and predicted pathways affected by the increase in starch intake, as well as by the phytogenic supplementation. Despite the differences in the microbial composition and metabolic potential of the different GIT niches, all showed similar changes toward carbohydrate metabolism. Metabolite measurement confirmed the high prevalence of glucose and volatile fatty acids (VFAs) in the rumen due to the increased substrate availability and metabolic activity of the microbiota. Families Prevotellaceae, Ruminococcaceae and Lachnospiraceae were found to be positively correlated with carbohydrate metabolism, with the latter two showing wide-ranging predicted metabolic capabilities. Phytogenic supplementation affected low abundant taxa and demonstrated the potential to prevent unwanted implications of feeding high-concentrate diet, such as reduction of microbial diversity. The inclusion of 50% concentrate in the diet caused a major shift in microbial composition and activity in the GIT of cattle. This study demonstrated the ability of microorganisms in various GIT niches to adjust differentially, yet rapidly, to changing dietary conditions, and revealed the potential beneficial effects of supplementation with a PFA during dietary adaptation.

Characterization of presence and activity of microRNAs in the rumen of cattle hints at possible host-microbiota cross-talk mechanism.

MicroRNAs (miRNAs), as important post-transcriptional regulators, are ubiquitous in various tissues. The aim of this exploratory study was to determine the presence of miRNAs in rumen fluid, and to investigate the possibility of miRNA-mediated cross-talk within the ruminal ecosystem. Rumen fluid samples from four cannulated Holstein cows were collected during two feeding regimes (forage and high-grain diet) and DNA and RNA were extracted for amplicon and small RNA sequencing. Epithelial biopsies were simultaneously collected to investigate the co-expression of miRNAs in papillae and rumen fluid. We identified 377 miRNAs in rumen fluid and 638 in rumen papillae, of which 373 were shared. Analysis of microbiota revealed 20 genera to be differentially abundant between the two feeding regimes, whereas no difference in miRNAs expression was detected. Correlations with at least one genus were found for 170 miRNAs, of which, 39 were highly significant (r > |0.7| and P < 0.01). Both hierarchical clustering of the correlation matrix and WGCNA analysis identified two main miRNA groups. Putative target and functional prediction analysis for the two groups revealed shared pathways with the predicted metabolic activities of the microbiota. Hence, our study supports the hypothesis of a cross-talk within the rumen at least partly mediated by miRNAs.

Effect of Duration of High-Grain Feeding on Chewing, Feeding Behavior, and Salivary Composition in Cows with or without a Phytogenic Feed Supplement.

Switching diets from forage to a high-grain (HG) diet increases the risk of rumen fermentation disorders in cattle. However, the effects of the duration of the HG feeding, after the diet switch, on animal behavior and health have received considerably less attention. This experiment primarily aimed to assess the effects of the duration of an HG diet on the chewing, eating, and lying behavior and salivation dynamics in a control group (CON) and a group of cows receiving a phytogenic feed supplement (TRT) at 0.04% (DM basis), which included L-menthol, thymol, eugenol, mint oil, and cloves powder. The experiment was a crossover design with nine non-lactating cows, and two experimental periods with an intermediate washout of four weeks. In each period, the cows were first fed a forage diet for a week to collect baseline measurements representing week 0; then, the diet was switched over a week to HG (65% concentrate), which was fed for four continuous weeks (week 1, week 2, week 3, and week 4 on an HG diet, respectively). The cows were divided in two groups of four and five animals and were randomly allocated to CON or TRT. The data analysis revealed that at the start of the HG feeding, the dry matter intake and the cows' number of lying bouts increased, but the eating time, rumination time, and meal frequency decreased, resulting in a greater eating rate. We also found that an advanced duration on an HG diet further decreased the rumination time, total chewing time, chewing index, and sorting in favor of short feed particles, with the lowest values in week 4. The feed bolus size increased but feed the ensalivation decreased in week 4 compared to week 0. The dietary switch increased salivary lysozyme activity, and the advanced duration on the HG diet increased salivary pH, but salivary phosphate decreased in weeks 1 and 2 on the HG diet. Supplementation with TRT increased sorting in favor of physically effective NDF (peNDF) in week 2 and increased salivary pH in week 4 on an HG diet. Overall, the negative effects of the HG diet in cattle are more pronounced during the initial stage of the HG feeding. However, several detrimental effects were exacerbated with the cows' advanced duration on feed, with host adaptive changes still observed after 3 and 4 weeks following the diet switch. The TRT mitigated some of the negative effects through the temporal improvement of the salivary properties and the intake of peNDF, which are known to modulate rumen fermentation.

Supplementing a Phytogenic Feed Additive Modulates the Risk of Subacute Rumen Acidosis, Rumen Fermentation and Systemic Inflammation in Cattle Fed Acidogenic Diets.

Feeding with high-concentrate diets increases the risk of subacute ruminal acidosis (SARA). This experiment was conducted to evaluate whether supplementing a phytogenic feed additive based on L-menthol, thymol, eugenol, mint oil (Mentha arvensis) and cloves powder (Syzygium aromaticum) (PHY) can amend the ruminal fermentation profile, modulate the risk of SARA and reduce inflammation in cattle. The experiment was designed as a crossover design with nine non-lactating Holstein cows, and was conducted in two experimental runs. In each run, cows were fed a 100% forage diet one week (wk 0), and were then transitioned stepwise over one week (0 to 65% concentrate, wk adapt.) to a high concentrate diet that was fed for 4 weeks. Animals were fed diets either with PHY or without (CON). The PHY group had an increased ruminal pH compared to CON, reduced time to pH < 5.8 in wk 3, which tended to decrease further in wk 4, reduced the ruminal concentration of D-lactate, and tended to decrease total lactate (wk 3). In wk 2, PHY increased acetate, butyrate, isobutyrate, isovalerate, and the acetate to propionate ratio compared to CON. Phytogenic supplementation reduced inflammation compared to CON in wk 3. Overall, PHY had beneficial effects on ruminal fermentation, reduced inflammation, and modulated the risk of SARA starting from wk 3 of supplementation.

Changes in fermentation profile of the reticulorumen and hindgut, and nutrient digestion in dry cows fed concentrate-rich diets supplemented with a phytogenic feed additive.

This study evaluated the effects of duration of high-concentrate feeding on ruminal and fecal fermentation profile, as well as selected systemic health biomarkers in nonlactating cows supplemented with or without a phytogenic feed additive (PHY). In addition, ruminal degradation kinetics and total-tract nutrient digestibility were evaluated when feeding either only forage or a high-concentrate diet. Nine nonlactating, cannulated Holstein cows were used in a crossover design. Each period included 1 wk of forage feeding (wk 0), diet transition, and 4 wk on the high-concentrate diet (1, 2, 3 and wk 4; 65% dry matter basis). Cows received PHY or not (control). Compared with wk 0, from wk 1 onward, cows on high concentrate showed greater reticular, ruminal, and fecal total volatile fatty acids (VFA), with a greater level of VFA in the rumen than in the hindgut. However, ruminal fermentation was modulated differently by PHY, which showed increased total VFA in wk 1 and increased butyrate in wk 2 in the particle-associated fluid of rumen. In the hindgut, PHY increased propionate in wk 3. Cows fed a high-concentrate diet from wk 1 and onward also showed greater ruminal lactate, as well as lower ruminal and fecal pH, independent of PHY. In addition, compared with cows in wk 1 on a high-concentrate diet, cows in wk 4 had a greater total VFA in free fluid of the rumen and lower fecal pH. Compared with cows at wk 0, cows at wk 1 on high concentrate onward showed greater serum amyloid A and greater activity of glutamate dehydrogenase. In contrast, the high-concentrate diet decreased in situ ruminal degradability of grass silage but increased degradability of corn grain as well as total-tract nutrient digestibility, with total-tract neutral detergent fiber digestibility being greater for cows on the PHY treatment. Overall, from the start of high-concentrate feeding, gut fermentation increased, but differently according to location or PHY, with a stronger build-up of VFA in the rumen compared with the hindgut. In addition, a longer duration on high concentrate exacerbated gut acidification. The enhancing effects of PHY on total VFA and butyrate in particle-associated fluid of the rumen suggest beneficial effects of PHY on particle-associated bacteria, likely contributing to the increased neutral detergent fiber digestibility. The greater production of ruminal butyrate with PHY may be beneficial for the host, given the health benefits of this acid, but more research is needed to elucidate the effects on gut microbiota and the effects of increased butyrate in nonlactating dairy cows.

Bovine rumen epithelial miRNA-mRNA dynamics reveals post-transcriptional regulation of gene expression upon transition to high-grain feeding and phytogenic supplementation.

The rumen epithelium has a pivotal role in nutrient uptake and host health. This study aimed to explore the role of microRNAs (miRNAs) in the epithelial transcriptome during diet transition from forage to high-grain feeding and the modulation through supplementation with a phytogenic feed additive. Rumen biopsies were collected from 9 ruminally-cannulated non-lactating Holstein cows fed a baseline forage diet (FD) and then transitioned to high-grain feeding (HG; 65% concentrate on a dry matter basis). Cows were randomly allocated into a control group (CON, n = 5) and a group supplemented with a phytogenic feed additive (PHY, n = 4). MiRNA and mRNA sequencing was performed in parallel and transcripts were analyzed for differential expression, pathway enrichment analysis, and miRNA-mRNA interaction networks. We identified 527 miRNAs shared by all samples of the rumen epithelium, from which, bta-miR-21-5p, bta-miR-143 and bta-miR-24-3p were the most expressed. Six miRNAs were differentially expressed between CON and PHY and 8 miRNAs between FD and HG feeding, which were mainly associated with fat metabolism. Transcriptome analysis identified 9481 differentially expressed genes (DEGs) between FD and HG, whereas PHY supplementation resulted in 5 DEGs. DEGs were mainly involved in epithelium development and morphogenesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with tricarboxylic acid and short chain fatty acid (SCFA) metabolism were enriched in DEGs between diets. MiRNA target prediction and anti-correlation analysis was used to construct networks and identify DEGs targeted by DE miRNAs responsive to diet or PHY. This study allowed the identification of potential miRNA regulation mechanisms of gene expression during transition from FD to HG feeding and phytogenic supplementation, evidencing a direct role of miRNAs in host responses to nutrition.

Supplementation With Phytogenic Compounds Modulates Salivation and Salivary Physico-Chemical Composition in Cattle Fed a High-Concentrate Diet.

Saliva facilitates feed ingestion, nutrient circulation, and represents an important pH buffer for ruminants, especially for cattle fed high-concentrate diets that promote rumen acidification. This experiment evaluated the short-term effects of nine phytogenic compounds on salivation, saliva physico-chemical composition as well as ingested feed boli characteristics in cattle. A total of nine ruminally cannulated Holstein cows were used. Each compound was tested in four of these cows as part of a high-concentrate meal (2.5 kg of total mixed ration in dry matter basis for 4 h) in low or high dose, and was compared to a control meal without compound. Saliva was sampled orally (unstimulated saliva) for physico-chemical composition analysis. Composition of the ingested saliva (stimulated saliva), salivation and feed boli characteristics were assessed from ingesta collected at the cardia during the first 30 min of the meal. Analysis of unstimulated saliva showed that supplementation with capsaicin and thyme oil increased buffer capacity, while supplementation with thymol, L-menthol and gentian root decreased saliva pH. In addition, supplementing angelica root decreased saliva osmolality. Regression analysis on unstimulated saliva showed negative associations between mucins and bicarbonate as well as with phosphate when garlic oil, thyme oil or angelica root was supplemented. Analysis of stimulated saliva demonstrated that supplementation with garlic oil increased phosphate concentration, thyme oil tended to increase osmolality, capsaicin and thymol increased buffer capacity, and ginger increased phosphate content. Furthermore, salivation rate increased with ginger and thymol, and tended to increase with garlic oil, capsaicin, L-menthol and mint oil. Feed ensalivation increased with capsaicin. A positive association was found between feed bolus size and salivation rate when any of the phytogenic compounds was supplemented. Overall, our results demonstrate positive short-term effects of several phytogenic compounds on unstimulated and stimulated saliva physico-chemical properties, salivation or feed boli characteristics. Thus, the phytogenic compounds enhancing salivary physico-chemical composition have the potential to contribute to maintain or improve ruminal health in cattle fed concentrate-rich rations.

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.