Endotoxin Translocation Is Increased in Broiler Chickens Fed a Fusarium Mycotoxin-Contaminated Diet.

Broiler chickens in livestock production face numerous challenges that can impact their health and welfare, including mycotoxin contamination and heat stress. In this study, we aimed to investigate the combined effects of two mycotoxins, deoxynivalenol (DON) and fumonisins (FBs), along with short-term heat stress conditions, on broiler gut health and endotoxin translocation. An experiment was conducted to assess the impacts of mycotoxin exposure on broilers, focusing on intestinal endotoxin activity, gene expression related to gut barrier function and inflammation, and the plasma concentration of the endotoxin marker 3-OH C14:0 either at thermoneutral conditions or short-term heat stress conditions. Independently of heat stress, broilers fed DON-contaminated diets exhibited reduced body weight gain during the starter phase (Day 1-12) compared to the control group, while broilers fed FB-contaminated diets experienced decreased body weight gain throughout the entire trial period (Day 1-24). Furthermore, under thermoneutral conditions, broilers fed DON-contaminated diets showed an increase in 3-OH C14:0 concentration in the plasma. Moreover, under heat stress conditions, the expression of genes related to gut barrier function (Claudin 5, Zonulin 1 and 2) and inflammation (Toll-like receptor 4, Interleukin-1 beta, Interleukin-6) was significantly affected by diets contaminated with mycotoxins, depending on the gut segment. This effect was particularly prominent in broilers fed diets contaminated with FBs. Notably, the plasma concentration of 3-OH C14:0 increased in broilers exposed to both DON- and FB-contaminated diets under heat stress conditions. These findings shed light on the intricate interactions between mycotoxins, heat stress, gut health, and endotoxin translocation in broiler chickens, highlighting the importance of understanding these interactions for the development of effective management strategies in livestock production to enhance broiler health and welfare.

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.

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.

A single, episodic event of unilateral/bilateral scrotal swelling in a group of adult boars at an Austrian boar stud.

BACKGROUND: Scrotal swelling is a clinical situation which can be caused by different aetiologies. In this case report, we describe a multi-week episode of unilateral and bilateral scrotal swelling in boars at an Austrian boar stud and its diagnostic work-up. CASE PRESENTATION: In the summer of 2020, the herd veterinarian of an Austrian boar stud reported that over a period of six weeks, five out of 70 boars presented with unilateral severe swelling of the left scrotum and three out of 70 boars with bilateral severe swelling of the left and moderate swelling of the right scrotum, respectively. A complete history was obtained and an on-site evaluation of the facility was done. Five boars were necropsied, and a variety of samples harvested for further diagnostic investigations. Infectious differential diagnoses associated with unilateral swelling of the scrotum or the testis were excluded through serological and tissue testing. In three of the five boars, histopathology revealed complete acute haemorrhagic necrosis of the left testis concurrent with strongly congested blood vessels. Review of the collected information with a group of experts in the field of boar stud management resulted with consensus that, most likely, trauma was the etiologic event causing the clinical signs and pathology. Coincident with discussion of implementing video recording cameras in the boar housing area, no further clinical cases followed. As this case occurred during the first lockdown of the COVID-19 pandemic, we propose that the distress and travelling restrictions may have contributed to frustration among boar stud workers, which was consequently expressed as misbehaviour against boars. CONCLUSIONS: Once all known infectious causes of unilateral swelling of the scrotum were excluded, a critical diagnostic work-up focused on non-infectious causes. Non-infectious causes, such as trauma, need to be carefully evaluated, as it may also include human misbehaviour against boars. Summarizing all findings of this case report, the authors hypothesize that a blunt trauma was the reason for the series of mainly unilateral swelling of the scrota of boars.

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.

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.

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.

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.

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.

Evaluation of a Dietary Grape Extract on Oxidative Status, Intestinal Morphology, Plasma Acute-Phase Proteins and Inflammation Parameters of Weaning Piglets at Various Points of Time.

Reports of the underlying mechanisms of dietary grape extract (GE) in overcoming weaning challenges in piglets have been partly inconsistent. Furthermore, evaluations of the effects of GE at weaning in comparison to those of widely used therapeutic antibiotics have been scarce. To explore the mode of action of GE in selected tissues and plasma, we evaluated gut morphology, antioxidant and inflammation indices. Accordingly, 180 weaning piglets were allocated to three treatment groups: negative control (NC), NC and antibiotic treatment for the first 5 days of the trial (positive control, PC), and NC and GE (entire trial). The villus surface was positively affected by GE and PC on day 27/28 of the trial in the jejunum and on day 55/56 of the trial in the ileum. In the colon, NC tended (p < 0.10) to increase crypt parameters compared to PC on day 55/56. The PC group tended (p < 0.10) to increase catalase activity in the ileum and decrease Cu/Zn-SOD activity in the jejunum, both compared to NC. There were no additional effects on antioxidant measurements of tissue and plasma, tissue gene expression, or plasma acute-phase proteins. In conclusion, GE supplementation beneficially affected the villus surface of the small intestine. However, these changes were not linked to the antioxidant and anti-inflammatory properties of GE.

Comparative Effects of Deoxynivalenol, Zearalenone and Its Modified Forms De-Epoxy-Deoxynivalenol and Hydrolyzed Zearalenone on Boar Semen In Vitro.

Deoxynivalenol (DON) and zearalenone (ZEN) are described as detrimental factors to sow and boar fertility. In comparison, literature reports on the impact of modified forms of DON and ZEN, such as de-epoxy-DON (DOM-1) and hydrolyzed ZEN (HZEN), on swine reproduction are scarce. The aim of our study was to compare the effects of DON, DOM-1, ZEN and HZEN on boar semen in vitro. To this end, pooled boar semen ejaculates from two adult boars were treated with either 50.6 µM DON, 62.8 µM ZEN or equimolar concentrations of DOM-1 and HZEN, respectively (dilution volume of v/v 0.7% DMSO in all cases). Effects on semen motility, morphology, viability, hypo-osmotic swelling test reaction and DNA integrity were investigated hourly up to four hours of incubation. DON negatively affected particular parameters evaluated with a computer-assisted sperm analysis system (CASA), such as immotile spermatozoa and progressive motile spermatozoa, whereas those effects were absent in the case of DOM-1 treatment. In contrast to HZEN, ZEN affected almost all CASA parameters. Furthermore, only ZEN decreased the proportion of viable spermatozoa and increased the proportion of spermatozoa with abnormalities. In conclusion, DON and ZEN negatively affected boar semen in vitro, whereas equimolar concentrations of DOM-1 and HZEN did not induce harmful effects.

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.

Establishment of an In Vitro Co-Culture Model of the Piglet Gut to Study Inflammatory Response and Barrier Integrity.

In intensive farming, piglets are exposed to various challenges that activate intestinal inflammatory processes, negatively affecting animal health and leading to economic losses. To study the role of the inflammatory response on epithelial barrier integrity, co-culture systems that mimic in vivo complexity are more and more preferred over cell monocultures. In this study, an in vitro gut co-culture model consisting of intestinal porcine epithelial cells and porcine peripheral blood mononuclear cells was established. The model provides an appropriate tool to study the role of the inflammatory response on epithelial barrier integrity and to screen for feed and food components, exerting beneficial effects on gut health. In the established model, inflammation-like reactions and damage of the epithelial barrier, indicated by a decrease of transepithelial electrical resistance, were elicited by activation of peripheral blood mononuclear cells via one of 3 stimuli: lipopolysaccharide, lipoteichoic acid, or concanavalin A. Two phytogenic substances that are commonly used as feed additives, licorice extract and oregano oil, have been shown to counteract the drop in transepithelial electrical resistance values in the gut co-culture model. The established co-culture model provides a powerful in vitro tool to study the role of intestinal inflammation on epithelial barrier integrity. As it consists of porcine epithelial and porcine blood cells it perfectly mimics in vivo conditions and imitates the inter-organ communication of the piglet gut. The developed model is useful to screen for nutritional components or drugs, having the potential to balance intestinal inflammation and strengthen the epithelial barrier integrity in piglets.

Supplementing a Clay Mineral-Based Feed Additive Modulated Fecal Microbiota Composition, Liver Health, and Lipid Serum Metabolome in Dairy Cows Fed Starch-Rich Diets.

Starch-rich diets are a commonly adopted strategy in order to sustain high milk yields in dairy cows. However, these diets are known to increase the risk of gut dysbiosis and related systemic health disorders. This study aimed to evaluate the effects of supplementing a clay mineral-based feed additive (CM; Mycofix® Plus, BIOMIN) on fecal microbiota structure, fecal short-chain fatty acid (SCFA) fermentation, serum metabolome, and liver health in primiparous (PP, n = 8) and multiparous (MP, n = 16) early-lactation Simmental cows (737 ± 90 kg of live body weight). Cows were randomly assigned to either a control or CM group (55 g per cow and day) and transitioned from a diet moderate in starch (26.3 ± 1.0%) to a high starch diet (32.0 ± 0.8%). Supplementation of CM reversed the decrease in bacterial diversity, richness, and evenness (p < 0.05) during high-starch diet, demonstrating that CM supplementation efficiently eased hindgut dysbiosis. The CM treatment reduced levels of Lactobacillus in PP cows during starch-rich feeding and elevated fecal pH, indicating a healthier hindgut milieu compared with that in control. Butyrate and propionate levels were modulated by CM supplementation, with butyrate being lower in CM-treated MP cows, whereas propionate was lower in MP but higher in PP cows. Supplementing CM during high-starch feeding increased the concentrations of the main primary bile salts and secondary bile acids in the serum and improved liver function in cows as indicated by reduced levels of glutamate dehydrogenase and γ-glutamyl-transferase, as well as higher serum albumin and triglyceride concentrations. These changes and those related to lipid serum metabolome were more pronounced in PP cows as also corroborated by relevance network analysis.

Potential of Grape Extract in Comparison with Therapeutic Dosage of Antibiotics in Weaning Piglets: Effects on Performance, Digestibility and Microbial Metabolites of the Ileum and Colon.

Enteric diseases in piglets, such as post-weaning diarrhea (PWD), often require antibiotic treatment of the entire litter. Grape polyphenols may help overcome PWD and thereby reduce the need for antibiotics. The potential of a grape extract (GE; continuous in-feed supplementation) on performance of weaning piglets, compared with both negative (NC; corn-based diet) and positive control (PC; NC + in-feed antibiotic (amoxicillin) in a therapeutic dosage for day 1-day 5 post weaning) was assessed. Apparent total tract digestibility (ATTD) and microbial metabolites were also evaluated on two sampling points (day 27/28 and day 55/56). We assigned 180 weaning piglets (6.9 ± 0.1 kg body weight (BW)) to 6 male and 6 female pens per treatment with 5 piglets each. Animals from PC showed higher BW on day 13 compared with NC and GE, and a tendency for higher BW on day 56 (p = 0.080) compared to NC. Furthermore, PC increased the average daily feed intake in the starter phase (day 1-day 13), and the average daily gain in the early grower phase (day 14-day 24). Overall, GE improved the ATTD at the same level as PC (ash, acid-hydrolyzed ether extract), or at a higher level than PC (dry matter, organic matter, gross energy, crude protein, P). There were no effects on microbial metabolites apart from minor trends for lactic acid and ammonia. Dietary inclusion of GE may have beneficial effects compared to therapeutic antibiotics, as frequently used at weaning.

Effect of Lipopolysaccharides (LPS) and Lipoteichoic Acid (LTA) on the Inflammatory Response in Rumen Epithelial Cells (REC) and the Impact of LPS on Claw Explants.

Endotoxins play a crucial role in ruminant health due to their deleterious effects on animal health. The study aimed to evaluate whether LPS and LTA can induce an inflammatory response in rumen epithelial cells. For this purpose, epithelial cells isolated from rumen tissue (REC) were stimulated with LPS and LTA for 1, 2, 4, and 24 h. Thereafter, the expression of selected genes of the LPS and LTA pathway and inflammatory response were evaluated. Furthermore, it was assessed whether LPS affects inflammatory response and structural integrity of claw explants. Therefore, claw explants were incubated with LPS for 4 h to assess the expression of selected genes and for 24 h to evaluate tissue integrity via separation force. LPS strongly affected the expression of genes related to inflammation (NFkB, TNF-α, IL1B, IL6, CXCL8, MMP9) in REC. LTA induced a delayed and weaker inflammatory response than LPS. In claw explants, LPS affected tissue integrity, as there was a concentration-dependent decrease of separation force. Incubation time had a strong effect on inflammatory genes in claw explants. Our data suggest that endotoxins can induce a local inflammatory response in the rumen epithelium. Furthermore, translocation of LPS might negatively impact claw health.

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.