Effects of Heat Stress on the Ruminal Epithelial Barrier of Dairy Cows Revealed by Micromorphological Observation and Transcriptomic Analysis

Heat stress (HS) alters the rumen fermentation of dairy cows thereby affecting the metabolism of rumen papillae and thus the epithelial barrier function. The aim of the present study was to investigate if HS damages the barrier function of ruminal epithelia. Eight multiparous Holstein dairy cows with rumen cannula were randomly equally allocated to two replicates (n = 4), with each replicate being subjected to heat stress or thermal neutrality and pair-feeding in four environmental chambers. Micromorphological observation showed HS aggravated the shedding of the corneum and destroyed the physical barrier of the ruminal epithelium to a certain extent. Transcriptomics analysis of the rumen papillae revealed pathways associated with DNA replication and repair and amino acid metabolism were perturbated, the biological processes including sister chromatid segregation, etc. were up-regulated by HS, while the MAPK and NF-kB cell signaling pathways were downregulated. However, no heat stress-specific change in the expression of tight junction protein or TLR4 signaling was found, suggesting that HS negatively affected the physical barrier of the ruminal epithelium to some extent but did not break the ruminal epithelium. Heat stress invoked mechanisms to maintain the integrity of the rumen epithelial barrier by upregulating the expression of heat shock protein and repairments in rumen papillae. The increase in amino acid metabolism in rumen papillae might affect the nutrient utilization of the whole body. The findings of this study may inform future research to better understand how heat stress affects the physiology and productivity of lactating cows and the development of mitigation strategies.

PSVIII-25 Transcriptional reprogramming in rumen epithelium during the developmental transition of pre-ruminant to the ruminant in cattle

The rumen is a critical organ mediating nutrient uptake and use in cattle. Healthy rumen development is essential to ensure animal feed efficiency. In this work, we present an analysis of transcriptomic dynamics in rumen epithelium during the transition from pre-rumination to rumination in cattle-fed hay or concentrated diets at weaning (eighteen Holstein bull calves, 3 X 6 groups). These two distinct phases of rumen development and function in cattle are tightly regulated by a series of signaling events and clusters of effectors on key pathways. Our analysis identifies putative signaling events and effectors. Gene activity shifts indicated the transcriptomic reprogramming required to induce developmental changes in ruminal epithelium and functional transitions. A principal component analysis distinguished the temporal expression patterns that clustered separately between pre- and post-weaning groups. A GO-term enrichment analysis reflected functional (physical and metabolic) development of ruminal epithelium and revealed the greatest number of DEGs were enriched in biological processes related to energy metabolism. Canonical pathway and upstream regulator analyses revealed transcription reprogramming with clusters of critical pathways and upstream regulators controlling functional and developmental transitions with no significant differences between hay- and concentrate-fed groups at weaning. The most highly activated transcription factors expressed during the weaning transition were PPARGC1A, INSR, NFE2L2, MYC, MYCN, and PPARA. Overall, the dietary shift from liquid to solid feeds prompted transcriptional reprogramming in rumen epithelial tissue reflecting critical nutrient-gene interactions occurring during the developmental progression of ruminant digestion.

Transcriptional Reprogramming in Rumen Epithelium during the Developmental Transition of Pre-Ruminant to the Ruminant in Cattle

We present an analysis of transcriptomic dynamics in rumen epithelium of 18 Holstein calves during the transition from pre-rumination to rumination in cattle-fed hay or concentrated diets at weaning. Three calves each were euthanized at 14 and 42 d of age to exemplify preweaning, and six calves each were provided diets of either milk replacer and grass hay or calf starter to introduce weaning. The two distinct phases of rumen development and function in cattle are tightly regulated by a series of signaling events and clusters of effectors on critical pathways. The dietary shift from liquid to solid feeds prompted the shifting of gene activity. The number of differentially expressed genes increased significantly after weaning. Bioinformatic analysis revealed gene activity shifts underline the functional transitions in the ruminal epithelium and signify the transcriptomic reprogramming. Gene ontogeny (GO) term enrichment shows extensively activated biological functions of differentially expressed genes in the ruminal epithelium after weaning were predominant metabolic functions. The transcriptomic reprogramming signifies a correlation between gene activity and changes in metabolism and energy production in the rumen epithelium, which occur at weaning when transitioning from glucose use to VFA use by epithelium during the weaning.

Direct Effect of Lipopolysaccharide and Histamine on Permeability Barrier of Rumen Epithelium

Background: Disruption of the ruminal epithelium barrier occurs during subacute ruminal acidosis due to low pH, hyper-osmolality, and increased concentrations of lipopolysaccharide and histamine in ruminal fluid. However, the individual roles of lipopolysaccharide and histamine in the process of ruminal epithelium barriers disruption are not clear. The objective of the present investigation was to evaluate the direct effect of lipopolysaccharide and histamine on barrier function of the ruminal epithelium. Results: Compared with control (CON), lipopolysaccharide (HIS) increased the short-circuit current (Isc) (88.2%, P = 0.0022), transepithelial conductance (Gt) (29.7%, P = 0.0564) and the permeability of fluorescein 5(6)-isothiocyanate (FITC) (1.04-fold, P = 0.0047) of ruminal epithelium. The apparent permeability of LPS was 1.81-fold higher than HIS (P = 0.0005). The mRNA abundance of OCLN in ruminal epithelium was decreased by HIS (1.1-fold, P = 0.0473). Conclusions: The results of the present study suggested that histamine plays a direct role in the disruption of ruminal epithelium barrier function while lipopolysaccharide without acidic pH has no significant effect on the permeability of rumen tissues.

Effects of Combined or Along VFA, pH, Lipopolysaccharide and Histamine on the Rumen Epithelial Permeability of Dairy Goats In Vitro

This study investigated whether concurrent presence of lipopolysaccharide (LPS) and histamine (HIS) have the potential to increase permeability of the ruminal epithelium at physiological pH and acidotic ruminal pH. Nine 2.5-year-old female lactating Saanen dairy goats (42.79 ± 5.61 kg of BW; Mean ± SD) were used as a ruminant model. ruminal epithelium of goats were collected and mounted in Ussing chambers on their mucosal side in different gradient buffer solutions (pH 7.4, 5.5 and 5.2) containing LPS (0, 30 and 60 KEU·mL-1) or HIS (0, 0.5 and 10 ng·mL-1). The rumen epithelial electrophysiological indexes, such as short-circuit (Isc), tissue conductance (Gt) and the permeability of marker molecules of different sizes (horseradish peroxidase, HRP and fluorescein 5(6)-isothiocyanate, FITC) from the mucosal to the serosal side, were measured. Both Isc and Gt were increased, accompanied by enhanced flux of FITC, with a decrease of mucosal pH (P < 0.05). The addition of LPS at mucosal pH 5.2 significantly increased Isc, Gt and FITC flux rates and decreased potential difference (PD) (P < 0.05). Additionally, the concurrent presence of LPS and HIS at both physiological and acidotic ruminal pH also significantly increased the permeability of ruminal epithelium asevidenced by increasing Isc, Gt and FITC flux rates and decreasing PD. In summary, our results have shown that concurrent presence of LPS 60 KEU‧mL-1 and HIS 10 ng‧mL-1 at mucosal pH 5.5 can increase the permeability of ruminal epithelium. The combination of low pH and both high LPS and HIS may increase vulnerability to aggravated rumen epithelial barrier dysfunction. © 2021 Friends Science Publishers

Thiamine Alleviates High-Concentrate-Diet-Induced Oxidative Stress, Apoptosis, and Protects the Rumen Epithelial Barrier Function in Goats

High-concentrate diets are continually used in ruminants to meet the needs of milk yield, which can lead to the occurrence of subacute rumen acidosis in ruminants. This study investigated the protective effects of dietary thiamine supplementation on the damage of the ruminal epithelium barrier function in goats fed a high-concentrate diet. Twenty-four healthy Boer goats (live weight of 35.62 ± 2.4 kg; age, 1 year) were randomly assigned into three treatments, with eight goats in each treatment, consuming one of three diets: a low-concentrate diet (CON; concentrate/forage, 30:70), a high-concentrate diet (HC; concentrate/forage, 70:30), or a high-concentrate diet with 200 mg of thiamine/kg of dry matter intake (HCT; concentrate/forage, 70:30) for 12 weeks. The additional dose of thiamine was based on our previous study wherein thiamine ameliorates inflammation. Compared with HC treatment, the HCT treatment had markedly higher concentrations of glutathione, superoxide dismutase, and glutathione peroxidase and total antioxidant capacity (P &lt; 0.05) in plasma and rumen epithelium. The results showed that the apoptosis index was lower (P &lt; 0.05) in the HCT treatment than in that of the HC treatment. Compared with the HC treatment, permeability and the electrophysiology parameter short circuit current for ruminal epithelial tissue were significantly decreased (P &lt; 0.05) in the HCT treatment. The immunohistochemical results showed that the expression distribution of tight junctions including claudin-1, claudin-4, occludin, and zonula occludin-1 (ZO-1) was greater (P &lt; 0.05) in the HCT treatments than in the HC treatment. The mRNA expression in the rumen epithelium of ZO-1, occludin, claudin-1, B-cell lymphoma/leukemia 2, nuclear factor erythroid-2 related factor 2 (Nrf2), superoxide dismutase 2 (SOD2), glutathione peroxidase 1, and the phase II metabolizing enzymes quinone oxidoreductase and heme oxygenase in the HCT group was significantly increased in comparison with the HC diet treatment (P &lt; 0.05), whereas the mRNA expression of caspase 3, caspase 8, caspase 9, bcl-2 associated X protein, lipopolysaccharide binding protein, toll-like receptor 4, nuclear factor kappa-B (NFκB), tumor necrosis factor alpha, interleukin-1β, interleukin, and tumor necrosis factor receptor-associated factor 6 decreased significantly in the HCT treatment (P &lt; 0.05). Compared with the HC treatment, the HCT diet significantly increased the protein expression of ZO-1, occludin, claudin-1, NQO1, HO-1, SOD2, serine/threonine kinase, p-Akt, Nrf2, and p-Nrf2; conversely, the expression of NFκB-related proteins p65 and pp65 was significantly decreased (P &lt; 0.05). In addition, thiamine relieved the damage on the ruminal epithelium caused by the HC diet. The results show that dietary thiamine supplementation improves the rumen epithelial barrier function by regulating Nrf2–NFκB signaling pathways during high-concentrate-diet feeding.

Effects of Flavonoids Extracted from Citrus aurantium on Performance, Behavior, and Rumen Gene Expression in Holstein Bulls Fed with High-Concentrate Diets in Pellet Form

Flavonoid supplementation may modify the behavior and rumen inflammatory response of fattening bulls, and this could be related to the concentrate presentation (mash or pellet) form. In the present study, 150 Holstein bulls (183.0 ± 7.53 kg BW and 137 ± 1.8 d of age) were randomly allocated to one of eight pens and assigned to control (C) or (BF) (Citrus aurantium, Bioflavex CA, HealthTech Bio Actives, Spain, 0.4 kg per ton of concentrate of Bioflavex CA, 20% naringin). Concentrate (pellet) intake was recorded daily, and BW and animal behavior fortnightly. Animals were slaughtered after 168 d of study, and ruminal epithelium samples were collected for gene expression analyses. Treatment did not affect animal performance; however, BF supplementation reduced agonistic interactions and oral non-nutritive behaviors and increased the time devoted to eating concentrate and ruminating activity (p < 0.05). The gene expression of some genes in the rumen epithelium was greater or tended to be greater in BF than C bulls (bitter taste receptor 16, cytokine IL-25, β-defensin; p < 0.10; pancreatic polypeptide receptor 1 and tumor necrosis factor alpha; p < 0.05). In conclusion, flavonoid supplementation modifies the expression of genes in the rumen epithelium that could be related to inflammation and animal behavior modulation.