Sex Differences in Rumen Fermentation and Microbiota of Tibetan Goat

Background: Gut microbiota play an important role in maintaining host metabolism, immune system and health, while sex, genotype, diet and health have certain effects on composition of gut microbiota. Therefore, in order to explore the sex differences in the structure and function of rumen microbiota in Tibetan goat, the study analyzed the sex differences in rumen fermentation parameters, rumen microbiota and the expression of genes related to VFAs transport in Tibetan goat. Results: The results showed that the content of acetic acid in the rumen of rams was significantly higher than that of ewes (P <0.05), and propionic acid and butyric acid were higher than that of ewes, but the difference was not significant (P >0.05); The expression of VFAs transport related genes DRA, AE2, MCT-1, NHE1, NHE2 in rumen epithelium of ewes was significantly higher than that of rams. Analysis of the composition and structure of rumen microbiota showed that there were significant sex differences in structure of rumen microbiota, and the abundance of rumen microbiota in ewes was higher than that in rams (P>0.05). At the phylum level, Firmicutes and Bacteroidetes were the dominant phyla of Tibetan goat, while Firmicutes was significantly higher in ewes than in rams (P<0.05). At the genus level, the relative abundance of Fibrobacter, Ruminococcus_1 and Pyramidobacter in ewes was significantly higher than that in rams (P<0.05). The function prediction results showed that Replication, recombination and repair, RNA processing and modification were mainly enriched in ewes (P<0.05). Conclusions: Correlation analysis revealed significant associations of some rumen microbiota with the fermentation product VFAs and VFAs transport-related genes. It is concluded that rams and ewes have a strong ability for fermenting and metabolizing when adapting to the plateau environment, which provides a certain sex reference basis for Tibetan goat adaptation to the plateau environment.

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.

The Rumen Microbiome and Its Metabolome Together With the Host Transcriptome Act to Regulate the Cold Season Adaptation Mechanism of Grazing Tibetan Sheep

BackgroundTibetan sheep are important ruminants on the Qinghai-Tibet Plateau. They can maintain a normal life and reproduce in harsh environments under extreme cold and low oxygen. However, the molecular and metabolic mechanisms underlying the adaptability of Tibetan sheep during the cold season are still unclear. Hence, we conducted a comprehensive analysis of rumen epithelial morphology, epithelial transcriptomics, microbiology and metabolomics in a Tibetan sheep model to understand the interaction between the rumen host and microbiota and their metabolites and to explore the potential regulatory mechanism of Tibetan sheep adaptability to the cold season of the plateau. ResultsMorphological analysis showed that the ruminal muscle layer thickness and nipple width of Tibetan sheep increased significantly during the cold season ( P <0.05), and the thickness of the stratum corneum, stratum granulosa and stratum spinous of the rumen epithelium increased significantly ( P <0.05). Transcriptomics analysis showed that the differential genes were primarily enriched in the PPAR signaling pathway (ko03320), legionellosis (ko05134), phagosome (ko04145), arginine and proline metabolism (ko00330), and metabolism of xenobiotics by cytochrome P450 (ko00980). Unique differential metabolites were identified in cold season, such as cynaroside A, sanguisorbin B and tryptophyl-valine, which were mainly enriched in arachidonic acid metabolism, arachidonic acid metabolism and linolenic acid metabolism pathways, and had certain correlation with microorganisms. Integrated transcriptome-metabolome-microbiome analysis showed that epithelial gene- GSTM3 expression was upregulated in the metabolism of xenobiotics by the cytochrome P450 pathway during the cold season, leading to the downregulation of some harmful metabolites; TLR5 gene expression was upregulated and CD14 gene expression was downregulated in the legionellosis pathway during the cold season. A large number of metabolites, such as glucosidic acid and vitamin A, were produced in the steroid hormone biosynthesis and retinol metabolism pathways. ConclusionThis study comprehensively described the interaction mechanism between the rumen host and microbes and their metabolites in grazing Tibetan sheep during the cold season. Under the stimulation of the cold plateau environment, the morphological structure of the rumen epithelium of Tibetan sheep undergoes adaptive changes. Rumen epithelial genes, microbiota and metabolites act together in some key pathways related to cold season adaptation.

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.

Establishment of a bovine rumen epithelial cell line

Rumen epithelium plays an essential role in absorption, transport, and metabolism of short-chain fatty acids, the main products of rumen fermentation, and in preventing microbes and other potentially harmful rumen contents from entering the systemic circulation. The objective of this study was to generate an immortal rumen epithelial cell line that can be used as a convenient model of rumen epithelial cells in vitro. We isolated primary rumen epithelial cells from a steer through trypsin digestion and transduced them with lentiviruses expressing the Simian Virus (SV) 40 T antigen. We cloned the transduced cells by limiting dilution. Western blotting analysis confirmed the expression of the SV40 T antigen in two single-cell clones. Cells from one clone, named bovine rumen epithelial clone 1 (BREC1), displayed a flat and squamous morphology in culture. RNA sequencing revealed that BREC1 cells expressed many markers of epithelial cells, including keratins, the epidermal growth factor receptor, and the short-chain fatty acid transporters monocarboxylic acid transporter 1 (MCT-1) and MCT-4. RNA sequencing revealed that BREC1 cells expressed key enzymes such as 3-hydroxymethyl-3-methylglutaryl-CoA lyase and 3-hydroxy-3-methylglutaryl-CoA synthase 1 involved in ketogenesis, a unique function of rumen epithelial cells. RNA sequencing also revealed the expression of genes encoding tight junctions, desmosomes, anchoring junctions, and polarized plasma membranes, structures typical of epithelial cells, in BREC1 cells. Cell proliferation assays indicated that BREC1 cells were similar to primary rumen epithelial cells in response to insulin-like growth factor 1, insulin, and butyrate. In conclusion, BREC1 is not only a convenient but an appropriate model for studying the factors and mechanisms that control proliferation, apoptosis, differentiation, nutrient transport, metabolism, and barrier function in rumen epithelium.

Niacin mitigates rumen epithelial damage in vivo by inhibiting rumen epithelial cell apoptosis on a high concentrate diet

To investigate the effects of niacin on rumen fermentation, rumen epithelial antioxidant activity, and rumen epithelial cell apoptosis on high concentrate (HC) diets, nine male Hu sheep were randomly divided into: low concentrate diet (LC; concentrate : forage (C:F) = 20:80, high concentrate diet (HC; C:F = 80:20), and HCN diet (HC diet + niacin at 800 mg/kg diet air-dry matter). Compared with the LC group, the HC group had a lower rumen pH, increased volatile fatty acids and lactic acid in the rumen, reduced activity of antioxidant enzymes and total antioxidant capacity, and increased malondialdehyde content in the rumen epithelium (P < 0.05). Rumen epithelial papilla morphology was decreased, and apoptosis-related indicators and serum inflammatory cytokines were increased in the HC group over the LC group (P < 0.05). Compared with the HC diet, the HCN diet increased rumen pH, rumen epithelium antioxidant capacity, and rumen epithelial papilla morphology, decreased rumen lactate content, serum inflammatory cytokines, and apoptosis-related indicators (P < 0.05). Therefore, adding 800 mg/kg niacin helped protect against rumen epithelial damage by avoiding drastic changes in the rumen environment and improved rumen epithelial antioxidant capacity to inhibit rumen epithelial cell apoptosis in sheep on a HC diet.

Oscillating dietary crude protein concentrations increase N retention of calves by affecting urea-N recycling and nitrogen metabolism of rumen bacteria and epithelium

The purpose of this study was to investigate the effects of oscillating crude protein (CP) concentration diet on the nitrogen utilization efficiency (NUE) of calves and determine its mechanism. Twelve Holstein calves were assigned randomly into static protein diet (SP, 149 g/kg CP) and oscillating protein diet (OP, 125 and 173 g/kg CP diets oscillated at 2-d intervals) groups. After 60 days of feeding, the weights of total stomach, rumen and omasum tended to increase in calves fed OP. The apparent crude fat digestibility, NUE and energy metabolism also increased. In terms of urea-N kinetics evaluated by urea-15N15N isotope labeling method, the urea-N production and that entry to gastrointestinal tended to increase, and urea-N reused for anabolism increased significantly in calves fed OP during the low protein phase. These data indicate that urea-N recycling contributed to improving NUE when dietary protein concentration was low. In addition, the differentially expressed genes in rumen epithelium and the rumen bacteria involved in protein and energy metabolism promoted the utilization of dietary protein in calves fed OP.

Effect of High Sulfur Diet on Rumen Fermentation, Microflora, and Epithelial Barrier Function in Steers

These experiments were conducted to evaluate the effect of excessive sulfur on rumen fermentation, microflora, and epithelial barrier function in steers through in vitro gas production and animal feeding experiments. Nine and four levels of sulfur addition were evaluated in in vitro ruminal fermentation and animal feeding experiment, respectively. The results showed that increasing the level of sulfur in substrates decreased the total gas and methane production linearly, while increasing the production of hydrogen sulfide gas (p < 0.01). Volatile fatty acid concentrations, especially that of butyric acid, were increased by extra sulfur (p < 0.01). Sulfur content in the diet had no significant effect (p > 0.05) on most of the rumen microbes, except for Desulfovibrio, one of the major sulfate-reducing bacteria (SRB) in the rumen, whose population increased by adding extra sulfur (p < 0.001). The changes in the morphology of rumen epithelium and thickening of the total epithelial layer were mainly attributed to the increase in the acanthosis cell layer and stratum basale (p < 0.05). Further, the relative expressions of two tight junction protein regulating genes, CLDN-1 and TJP1, were reduced (p < 0.05). Excessive sulfur in the diet can change the type of rumen fermentation, sulfate metabolism and SRB population, and the rumen epithelial barrier function. The results of this study demonstrated that sulfur can be used as a methane inhibitor with the mechanism that SRB competitively used protons to produce hydrogen sulfide. However, a higher level of sulfur in the diet could increase the inflammatory reaction of the rumen epithelium which may affect nutrient absorption.

Effect of Tea Tree Oil on the Expression of Genes Involved in the Innate Immune System in Goat Rumen Epithelial Cells

In subacute rumen acidosis (SARA), the rumen epithelium is frequently attacked by endotoxin (LPS), which is caused by the lysis of dead Gram-negative bacteria. However, the rumen epithelium innate immune system can actively respond to the infection. Previous studies have demonstrated that tea tree oil (TTO) has good bactericidal and anti-inflammatory effects. Therefore, the aim of this study was to investigate the effect of TTO on the expression of genes involved in the inflammatory cytokines in goat rumen epithelial cells (GRECs) triggered by LPS. Our study shows that rumen epithelial cells isolated from goat rumen tissue can be cultured in vitro in 0.25% trypsin for a long time. These cells were identified as epithelial cells by the expression of cytokeratin 18, monocarboxylate transporter 4 (MCT4), Na[+]/H[+] hydrogen exchanger 1 (NHE1), putative anion transporter 1 (PAT1), vH+ ATPase B subunit (vH+ ATPase), and anion exchanger 2 (AE2). The mRNA expression of IL-1β, IL-6, TNF-α, TLR-2, NF-κB, CXCL6 and CXCL8 genes was significantly increased when LPS was used compared to untreated controls. In addition, mRNA expression of IL-1β, IL-6, TNF-α, TLR-2, NF-κB, CXCL8, CXCL6 and interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) genes was also significantly higher in the LPS group compared to the 0.05% TTO group. However, the expression of IL-1β, IL-6, TNF-α, TLR-2, CXCL6 and IFIT3 genes was significantly lower in the LPS and 0.05% TTO group compared to the 1 μg/mL LPS group. These results suggest that TTO can inhibit LPS-induced inflammatory cytokines expression in GRECs.

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.