Impact of a Saccharomyces cerevisiae fermentation product during an intestinal barrier challenge in lactating Holstein cows on ileal microbiota and markers of tissue structure and immunity.

Feeding a Saccharomyces cerevisiae fermentation product (SCFP; NutriTek, Diamond V, Cedar Rapids, IA) during periods of metabolic stress is beneficial to the health of dairy cows partially through its effect on the gut microbiota. Whether SCFP alters the ileal microbiota in lactating cows during intestinal challenges induced by feed restriction (FR) is not known. We used 16S rRNA sequencing to assess if feeding SCFP during FR to induce gut barrier dysfunction alters microbiota profiles in the ileum. The mRNA abundance of key genes associated with tissue structures and immunity was also detected. Multiparous cows (97.1 ±â€…7.6 days in milk (DIM); n = 7 per treatment) fed a control diet or the control plus 19 g/d NutriTek for 9 wk were subjected to an FR challenge for 5 d, during which they were fed 40% of their ad libitum intake from the 7 d before FR. All cows were slaughtered at the end of FR. DNA extracted from ileal digesta was subjected to PacBio Full-Length 16S rRNA gene sequencing. High-quality amplicon sequence analyses were performed with Targeted Amplicon Diversity Analysis and MicrobiomeAnalyst. Functional analysis was performed and analyzed using PICRUSt and STAMP. Feeding SCFP did not (P > 0.05) alter dry matter intake, milk yield, or milk components during FR. In addition, SCFP supplementation tended (P = 0.07) to increase the relative abundance of Proteobacteria and Bifidobacterium animalis. Compared with controls, feeding SCFP increased the relative abundance of Lactobacillales (P = 0.03). Gluconokinase, oligosaccharide reducing-end xylanase, and 3-hydroxy acid dehydrogenase were among the enzymes overrepresented (P < 0.05) in response to feeding SCFP. Cows fed SCFP had a lower representation of adenosylcobalamin biosynthesis I (early cobalt insertion) and pyrimidine deoxyribonucleotides de novo biosynthesis III (P < 0.05). Subsets of the Firmicutes genus, Bacteroidota phylum, and Treponema genus were correlated with the mRNA abundance of genes associated with ileal integrity (GCNT3, GALNT5, B3GNT3, FN1, ITGA2, LAMB2) and inflammation (AOX1, GPX8, CXCL12, CXCL14, CCL4, SAA3). Our data indicated that the moderate FR induced dysfunction of the ileal microbiome, but feeding SCFP increased the abundance of some beneficial gut probiotic bacteria and other species related to tissue structures and immunity.

Stressors, including limited access to feed, heat stress, transportation, and disease are factors that reduce integrity of the gut epithelial barrier in livestock. Feeding Saccharomyces cerevisiae fermentation products (SCFP) mitigated immunological, aflatoxin, and subclinical mastitis challenges, heat stress, and grain-based subacute ruminal acidosis indicating it also could alleviate gut damage. Microbiota profiling of ileal epithelium using 16S rRNA sequencing and bioinformatics revealed that Lactobacillales and Animalis abundance was greater in cows fed SCFP versus controls during a 5-d feed restriction to induce intestinal dysfunction. Some genera of Firmicutes, Bacteroidota phylum, and Treponema genus were correlated with mRNA abundance of genes associated with integrity and inflammation of ileal epithelium. Thus, feeding SCFP can increase the abundance of beneficial bacteria during a gut challenge.

Alterations in ileal transcriptomics during an intestinal barrier challenge in lactating Holstein cows fed a Saccharomyces cerevisiae fermentation product identify potential regulatory processes.

Stressors such as lack of access to feed, hot temperatures, transportation, and pen changes can cause impairment of ruminal and intestinal barrier function, also known as "leaky gut". Despite the known benefits of some nutritional approaches during periods of stress, little is understood regarding the underlying mechanisms, especially in dairy cows. We evaluated the effect of feeding a Saccharomyces cerevisiae fermentation product (SCFP; NutriTek, Diamond V, Cedar Rapids, IA) on the ileal transcriptome in response to feed restriction (FR), an established model to induce intestinal barrier dysfunction. Multiparous cows [97.1 ±â€…7.6 days in milk (DIM); n = 5/group] fed a control diet or control plus 19 g/d SCFP for 9 wk were subjected to an FR challenge for 5 d during which they were fed 40% of their ad libitum intake from the 7 d before FR. All cows were slaughtered at the end of FR, and ileal scrapping RNA was used for RNAseq (NovaSeq 6000, 100 bp read length). Statistical analysis was performed in R and bioinformatics using the KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO databases. One thousand six hundred and ninety-six differentially expressed genes (DEG; FDR-adjusted P ≤ 0.10) were detected in SCFP vs. control, with 451 upregulated and 1,245 downregulated. "Mucin type O-glycan biosynthesis" was the top downregulated KEGG pathway due to downregulation of genes catalyzing glycosylation of mucins (GCNT3, GALNT5, B3GNT3, GALNT18, and GALNT14). An overall downregulation of cell and tissue structure genes (e.g., extracellular matrix proteins) associated with collagen (COL6A1, COL1A1, COL4A1, COL1A2, and COL6A2), laminin (LAMB2), and integrins (ITGA8, ITGA2, and ITGA5) also were detected with SCFP. A subset of DEG enriched in the GO term "extracellular exosome" and "extracellular space". Chemokines within "Cytokine-cytokine receptor interaction pathways" such as CCL16, CCL21, CCL14, CXCL12, and CXCL14 were downregulated by SCFP. The "Glutathione metabolism" pathway was upregulated by SCFP, including GSTA1 and RRM2B among the top upregulated genes, and GSTM1 and GPX8 as top downregulated genes. There were 9 homeobox transcription factors among the top 50 predicted transcription factors using the RNAseq DEG dataset, underscoring the importance of cell differentiation as a potential target of dietary SCFP. Taken together, SCFP downregulated immune-, ECM-, and mucin synthesis-related genes during FR. Homeobox transcription factors appear important for the transcriptional response of SCFP.

Stressors such as lack of access to feed, hot temperatures, transportation, and disease contribute to diminished gut epithelial barrier integrity in livestock. RNA-sequencing technology and bioinformatics were used to evaluate genome-wide mRNA abundance profiles in ileal tissue from dairy cows fed Saccharomyces cerevisiae fermentation product (SCFP) or an unsupplemented control diet during an intestinal challenge induced by feed restriction. Molecular responses were characterized according to metabolic pathways and other biological categories. Genes associated with "Mucin type O-glycan biosynthesis" and "Extracellular matrix-receptor interaction" were downregulated due to SCFP relative to controls. Alterations in cytokine and chemokine mRNA profiles induced by SCFP underscored differences in tissue immune response. Overall, SCFP altered the transcriptome of ileal tissue damaged by feed restriction.

Taraxasterol alleviates fatty acid-induced lipid deposition in calf hepatocytes by decreasing ROS production and endoplasmic reticulum stress.

Increased concentrations of free fatty acids (FFAs) induce reactive oxygen species (ROSs) generation and endoplasmic reticulum (ER) stress, thus, increasing the risk of fatty liver in dairy cows during the periparturient period. In non-ruminants, Taraxasterol (Tara; a pentacyclic triterpenoid found in medicinal plants) plays an important role in anti-inflammatory and antioxidant reactions. Whether Tara can alleviate or prevent fatty liver in ruminants is unknown. We addressed whether Tara supply could dampen lipid accumulation, ROSs production, and ER stress caused by FFAs in calf hepatocytes. Primary calf hepatocytes were isolated from five healthy calves (1 d old, female, 30-40 kg, fasting, rectal temperature 38.7-39.7 °C). In the first experiment, hepatocytes were incubated with various concentrations of Tara (2.5, 5, and 10 µg/mL) for 12 h prior to the 1.2-mM FFAs challenge. Results indicated that the level of ROSs was lowest with 5 µg/mL Tara. Thus, to further characterize the molecular mechanisms whereby Tara protects from FFAs-induced lipid deposition in calf hepatocytes, we performed incubations with 5 µg/mL Tara for 12 h prior to a 1.2-mM FFAs challenge for an additional 12 h. Results indicated that 1.2-mM FFAs challenge increased mitochondrial membrane potential (MMP), enhanced expression of proteins and mRNA associated with ER stress (PERK, IRE1, GRP78, ATF6, and CHOP) and fatty acid synthesis (FASN, ACC1, and SREBP-1c), and ultimately led to increased lipid droplet synthesis. In contrast, Tara treatment alleviated these negative effects after 1.2-mM FFAs challenge. To determine whether Tara protects against FFAs-induced lipid droplet synthesis by alleviating oxidative stress, hepatocytes were treated with 5 µg/mL Tara for 22 h prior to H2O2 (440 µM) challenge for 2 h. Compared with H2O2 treatment alone, results revealed a marked decrease in ROSs, MMP, and protein abundance of ER stress (GRP78, ATF6, and CHOP) and lipid droplet synthesis in response to Tara prior to H2O2 challenge. Data suggested that the increase in mitochondrial ROSs production contributes to lipid accumulation in calf hepatocytes. Collectively, our in vitro data indicate that Tara alleviates fatty acid-induced lipid deposition. Further research is warranted to ascertain that Tara can be helpful in the therapeutic management of early lactating cows to control or alleviate excessive hepatic lipid deposition.

Fatty liver is a common occurrence in the early postpartum period, partly due to the large influx of fatty acids into the liver during adipose tissue lipolysis. Because there is a linkage between fatty acid metabolism, oxidative stress, and lipid deposition in hepatocytes of nonruminant animals, we evaluated the potential therapeutic roles of Taraxasterol on reactive oxygen species and endoplasmic reticulum (ER) stress in vitro. This compound found in medicinal plants alleviated oxidative and ER stress and reduced lipid accumulation. Thus, it may represent a novel therapeutic tool for the management of dairy cows around parturition.

Feeding a Saccharomyces cerevisiae fermentation product before and during a feed restriction challenge on milk production, plasma biomarkers, and immune function in Holstein cows.

Periods of decreased feed intake may disrupt function of the intestinal barrier. Feeding NutriTek® (NTK; Diamond V, Cedar Rapids, IA), a postbiotic from S. cerevisiae fermentation (SCFP), improved health and supported anti-inflammatory functions. We investigated the effects of feeding NTK to cows before and during a period of feed restriction (FR) designed to model periods of intestinal barrier dysfunction. In total, 16 multiparous cows (97.1 ± 7.6 DIM; n = 8/group) were fed a control diet (CON) or CON plus 19 g/d NTK for 9 wk (Phase 1; P1) and then were subjected to an FR challenge for 5 d, during which they were fed 40% of their ad libitum intake from the 7 d prior to FR. Milk yield (MY) and DMI were collected daily. During FR, milk was collected daily for composition, blood daily to measure plasma biomarkers and to measure monocyte and neutrophil phagocytosis and oxidative burst on d 1, 3, and 5. Data were analyzed using a mixed model in SAS 9.4. All data were subjected to repeated measures ANOVA. Dietary treatment (TRT), Day, and their interaction (TRT × Day) were considered as fixed effects and cow as the random effect. For analysis of P1, data collected during a 7-d adaptation phase were used as a covariate. During P1, NTK cows tended to have greater DMI and had greater fat, ECM and FCM yields, and feed efficiency (ECM/DMI and FCM/DMI). Protein yield tended to be greater in NTK compared with CON cows. A tendency for greater monocyte phagocytosis was detected with NTK. However, during FR, feeding NTK led to lower MY and lactose yield and tended to lower solids percentage. While NTK cows tended to have reduced neutrophil oxidative burst than CON cows during FR (NTK: 26.20%, CON: 36.93%), there was no difference in phagocytosis (NTK: 7.92%, CON: 6.31%). Plasma biomarkers of energy metabolism, liver function, inflammation, and oxidative stress during the FR period did not differ. Overall, results suggested that feeding NTK increased the yield of FCM, ECM, feed efficiency and milk components prior to FR.

Postbiotic fermentation products have the potential to improve health and support anti-inflammatory functions when fed to lactating dairy cows. Since dairy cows experience disruptions of the intestinal barrier function at various stages of their life, for example, the transition into lactation, we sought to investigate potential beneficial effects of feeding a Saccharomyces cerevisiae fermentation (NTK) before and during a period of feed restriction to challenge gut function. Although feeding NTK increased yield of energy-corrected milk and feed efficiency prior to feed restriction (FR), it had no effect on production or plasma indices of metabolism, inflammation, and liver function during a period of abrupt FR to 40% of baseline feed intake.

Bacillus subtilis Produces Amino Acids to Stimulate Protein Synthesis in Ruminal Tissue Explants via the Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Beta-Serine/Threonine Kinase-Mammalian Target of Rapamycin Complex 1 Pathway.

Background: Bacillus subtilis is a probiotic strain that is widely used as a feed supplement for ruminants. In this study, one B. subtilis strain isolated from the ruminal fluid of Holstein dairy cows was used for an ex vivo study with ruminal tissue explants. The main goal was to assess the potential endosymbiotic links between B. subtilis and the ruminal epithelium using molecular analyses and amino acid profiling. The explant culture protocol was first optimized to determine the ideal conditions in terms of tissue viability before performing the actual experiments involving active and inactive bacteria with or without protein synthesis inhibitors, such as LY294002 (phosphatidylinositol 3-kinase inhibitor) or rapamycin [mammalian target of rapamycin (mTOR) inhibitor]. Results: The mRNA levels of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta (PIK3CB), serine/threonine kinase (AKT), mTOR, P70S6K1, and eukaryotic translation initiation factor 4E binding protein 1 were the highest (p < 0.01), while those of programmed cell death 4 were the lowest when the tissue was incubated with 107 of B. subtilis. Compared with the inactivated bacteria, the expression levels of PIK3CB and AKT, and overall changes in mTOR and P70S6K1 were greater in rumen explants with living bacteria (p < 0.05). With an increase in B. subtilis concentration, the trends of protein and corresponding gene changes were consistent. There were differences in the concentrations of individual amino acids in the supernatants of living and inactivated bacterial culture groups, with most amino acids enriched in pathways, such as aminoacyl tRNA biosynthesis, cyanoamino acid metabolism, monobactam biosynthesis, or glycine, serine, and threonine metabolism. The addition of psilocybin upregulated the expression levels of PIK3CB and AKT. A significant decrease (p < 0.05) in PIK3CB and mTOR protein expression levels was detected after the addition of LY294002 and rapamycin. In addition, These responses were associated with the downregulation (p < 0.05) of AKT and P70S6K protein expression levels. Conclusions: We confirmed that the in vivo ruminal tissue culture system is a suitable model for studying probiotic-induced alterations in tissue function. As such, this study provides a means for future mechanistic studies related to microbial regulation and the dietary supply of proteins. In addition, living and inactivated B. subtilis can promote protein synthesis in ruminal tissue explants by altering the expression levels of related factors in the PIK3CB-AKT-mTORC1 pathway, which could further aid in optimizing the feed efficiency and increasing the use of inactivated bacteria as additives in dairy cow farming.