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Feeding a Saccharomyces cerevisiaefermentation 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 ± 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 Treponemagenus 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.Feeding a Saccharomyces cerevisiaefermentation product during intestinal dysfunction alters microbiota associated with the structure and immune response of the ileum in a positive fashion.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 cerevisiaefermentation 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 Lactobacillalesand Animalisabundance was greater in cows fed SCFP versus controls during a 5-d feed restriction to induce intestinal dysfunction. Some genera of Firmicutes, Bacteroidotaphylum, and Treponemagenus 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.

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 cerevisiaefermentation 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 ± 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 CXCL14were downregulated by SCFP. The “Glutathione metabolism” pathway was upregulated by SCFP, including GSTA1and RRM2Bamong the top upregulated genes, and GSTM1and GPX8as 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.Feeding a Saccharomyces cerevisiaefermentation product to lactating cows reduced immune-related gene transcription in the ileum during feed restriction.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 cerevisiaefermentation 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.

Physiological and environmental stresses such as the transition into lactation and heat load contribute to gastrointestinal tract (GIT) dysfunction. The nonruminant gastrointestinal tract has mechanisms to cope with pro-oxidant and pro-inflammatory stressors arising from the gut lumen or within intestinal cells. One-carbon metabolism (OCM) contributes to anti-oxidant capacity via the production of glutathione (GSH) and taurine, and the synthesis of phospholipid, creatine, and the osmolyte glycinebetaine among others. A multipronged approach was used to assess the biological relevance of OCM and closely-related pathways on GIT function in dairy cows. Ruminal papillae (Rum) and scrapings from duodenum (Duo), jejunum (Jej), and ileum (Ile) were collected at slaughter from eight multiparous Holstein cows averaging 128 ± 12 d in milk and producing 39 ± 5 kg/d. A MIXED model ANOVA with preplanned orthogonal contrasts was used for statistical analysis. Methionine adenosyl transferase 1 activity (MAT) was ~10-fold greater (P< 0.01) and cystathionine β-synthase activity doubled in Rum vs. small intestine. Total glutathione peroxidase (GPX) activity was greatest (P= 0.03) in Ile, but similar to Rum. Activity and mRNA abundance of betaine-homocysteine S-methyltransferase were undetectable. There was a 2.5-fold greater protein abundance of GPX1 (P< 0.01) and a ~2-fold greater abundance of GPX3 (P< 0.01) in Rum vs. small intestine. Among the various amino acids (AA) with roles in OCM or closely-related pathways (e.g. creatine synthesis), concentrations of arginine, aspartate, glutamine, methionine, and serine were lower (P< 0.01) in Rum vs. small intestine. Unlike AA, concentrations of OCM-related intermediates S-5ʹ-adenosyl-homocysteine (SAH), glycinebetaine, carnitine, creatine (CRE), and cysteinesulfinic acid were greater (P< 0.01) while taurine was lower in Rum vs. small intestine. Intermediates of the folate cycle were undetectable. The fact that S-adenosylmethionine (SAM) was undetectable while MAT activity and SAH were greater in Rum suggested that availability of SAM (a methyl donor) is a key determinant of flux through the folate and methionine cycles in the GIT. Except for adenosine, concentrations of glutamate, glycine, α-ketoglutarate, hypotaurine, and GSH were lowest in Ile. Together, the data underscored unique differences in activity of one-carbon metabolism and related pathways across sections of the GIT.Anti-oxidant mechanisms associated with one-carbon metabolism are more active in ruminal vs. small intestinal tissue of lactating cows.The gastrointestinal tract serves a number of essential functions in the animal and exposure to physiological and environmental stressors can lead to disruption of its barrier function and compromise nutrient absorption. In nonruminants, mechanisms to cope with pro-oxidant and pro-inflammatory stressors are essential for maintaining gut function. One-carbon metabolism contributes to anti-oxidant capacity via the production of glutathione and taurine, synthesis of phospholipids, energy-producing compounds, and the osmolyte glycinebetaine among others. A multipronged approach was used to assess the biological relevance of one-carbon metabolism and closely-related pathways in the rumen and small intestine of lactating dairy cows. Enzyme activities, mRNA and protein abundance, and metabolite profiling revealed unique patterns in the rumen versus small intestine. Methyl donor synthesis, transsulfuration, glutathione synthesis, and glutathione peroxidase activity are active mechanisms in ruminal tissue. Research targeting the alteration of these pathways through specific nutrients during stressful periods such as the transition into lactation, weaning, and heat load is warranted.