We tested the hypothesis that the proliferative effects of intraruminal butyrate infusions on the ruminal epithelium are linked to upregulation in cyclin D1 (CCND1), the cyclin-dependent kinase 4 (CDK4), and their possible association with enhanced absorption of short-chain fatty acids (SCFA). Goats (n=23) in 2 experiments (Exp.) were fed 200 g/d concentrate and hay ad libitum. In Exp. 1, goats received an intraruminal infusion of sodium butyrate at 0.3 (group B, n=8) or 0 (group C, n=7) g/kg of body weight (BW) per day before morning feeding for 28 d and were slaughtered 8 h after the butyrate infusion. In Exp. 2, goats (n=8) received butyrate infusion and feeding as in Exp. 1. On d 28, epithelial samples were biopsied from the antrium ruminis at 0, 3, and 7 h after the last butyrate infusion. In Exp. 1, the ruminal molar proportional concentration of butyrate increased in group B by about 110% after butyrate infusion and remained elevated for 1.5 h; thereafter, it gradually returned to the baseline (preinfusion) level. In group C, the molar proportional concentration of butyrate was unchanged over the time points. The length and width of papillae increased in B compared with C; this was associated with increased numbers of cells and cell layers in the epithelial strata and an increase in the surface area of 82%. The mRNA expression of CCND1 increased transiently at 3 h but returned to the preinfusion level at 7 h following butyrate infusion in Exp. 2. However, it did not differ between B and C in Exp. 1, in which the ruminal epithelium was sampled at 8 h after butyrate infusion. The mRNA expression of the monocarboxylate transporter MCT4, but not MCT1, was stably upregulated in B compared with C. The estimated absorption rate of total SCFA (%/h) increased in B compared with C. We conclude that transient increases in cyclin D1 transcription contribute to butyrate-induced papillae growth and subsequently to the increased absorption of SCFA in the ruminal epithelium of goats.

Currently, the mechanism(s) responsible for the regulation of urea transporter B (UT-B) expression levels in the epithelium of the rumen remain unclear. We hypothesized that rumen fermentation products affect ruminal UT-B expression. Therefore, the effects of short-chain fatty acids (SCFA), pH, ammonia, and urea on mRNA and protein levels of UT-B were assayed in primary rumen epithelial cell cultures and in rumen epithelium obtained from intact goats. In vitro, SCFA and acidic pH were found to synergetically stimulate both mRNA and protein expression of UT-B, whereas NH 4Cl decreased mRNA and protein levels of UT-B at pH 6.8. Treatment with urea increased both levels at pH 7.4. When goats received a diet rich in nitrogen (N) and nonfiber carbohydrates (NFC), their rumen epithelium had higher levels of UT-B, and the rumen contained higher concentrations of SCFA and NH 3-N with a lower pH. An increase in plasma urea-N concentration was also observed compared with the plasma of the goats that received a diet low in N and NFC. In a second feeding trial, goats that received a NFC-rich, but isonitrogenous, diet had higher mRNA and protein levels of UT-B, and higher levels of G proteincoupled receptor (GPR) 41 and GPR4, in their rumen epithelium. The ruminal concentrations of SCFA and NH 3-N also increased, while a lower pH was detected. In contrast, the serum urea-N concentrations remained unchanged. These data indicate that ruminal SCFA and pH are key factors, via GPR4 and GPR41, in the dietary regulation of UT-B expression, and they have priority over changes in plasma urea.SCFA and pH; UT-B expression; GPR41; GPR4; rumen epithelial cells; diet RECYCLING OF UREA TO THE MAMMALIAN gut accounts for 20 -30% of liver-synthesized urea that is present in humans (17). In contrast, these levels vary from 10 to 90% in ruminants (21) with feeding strategy (21) and intake of fermentable carbohydrates (22). These characteristics make the ruminant forestomach, especially the rumen, an excellent model for studies of urea entry and regulation in the gut. Urea transport across the rumen epithelium is generally accepted to occur down a concentration gradient from blood to lumen by diffusion through transport proteins, such as urea transporter B (UT-B), or possibly aquaporins. In our laboratory's recent studies, rumen fermentation products, including short-chain fatty acids (SCFA) and ammonia, as well as pH, were found to rapidly modulate UT-B activity (1, 34). It is possible that multiple mechanisms mediate regulation of urea transport, although these mechanisms are not fully characterized. UT-B has been found in many tissues and species, including human and rodent colon tissues, in erythrocytes and kidney tissue, as well as in ovine and bovine rumen (52). In bovine tissues, the UT-B protein exists as a multimer (30). Previous studies (21, 39, 41) have shown that changes in dietary nitrogen (N) content causes significant changes in serum urea-N (BUN) concentrations and the entry of urea into the gut. However, a correlation between N ...

Short-chain fatty acids (SCFA) regulate cell proliferation and cell apoptosis in gastrointestinal tissue in vitro and in vivo. We have tested the hypothesis that a medium-concentrate intake induces mRNA abundance alterations of genes involved in cell proliferation and cell apoptosis in the rumen epithelium of goats, and that these changes in mRNA abundance are related to ruminal SCFA concentration and ruminal pH. Goats (n=16) were randomly allocated to 2 groups and fed either a low-concentrate (LC) diet (10% concentrate; n=8) or a medium-concentrate (MC) diet (35% concentrate; n=8) in 2 equal portions daily. The individually housed goats were fed separately with their respective diet for 3wk and were slaughtered 6h after the morning feed on d 22. In vivo, goats receiving the MC treatment exhibited a greater ruminal SCFA concentration (73.7mM) compared with those receiving the LC treatment (53.2mM), and the pH decreased from 6.9 to 6.5. The expression of proliferative genes of cyclin A, cyclin B1, cyclin D1, cyclin E1, CDK1, CDK2, CDK4, and CDK6 mRNA in the MC group was enhanced. The gene expression of apoptosis genes (caspase 3, caspase 8, caspase 9, p53, and Bax) was significantly higher, and the ratio of Bcl-2 to Bax (Bcl-2/Bax) expression was lower in the MC group than in the LC group. The same trend was observed in the population of apoptotic cells analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. The cell density in the stratum germinativum of the MC group was significantly increased compared with that in the LC group. During primary culture of rumen epithelial cells, SCFA or pH treatment alone of the culture medium had significant effects on the expression of most of the genes tested in the present study. Furthermore, SCFA and pH exerted combined effects on the expression of cyclin A, cyclin B1, cyclin E1, CDK6, p53, Bcl-2, and Bcl-2/Bax. Thus, the MC diet induces alteration of gene expression of the genes that regulate both cell proliferation and apoptosis. These genes are regulated by combined effect of ruminal SCFA and ruminal pH.

In our previous study, we demonstrated that butyrate induced ruminal epithelial growth through cyclin D1 upregulation. Here, we investigated the influence of butyrate on the expression of genes associated with cell cycle and apoptosis in rumen epithelium. Goats (n = 24) were given an intra ruminal infusion of sodium butyrate at 0.3 (group B, n = 12) or 0 (group A, n = 12) g/kg of body weight (BW) per day before morning feeding for 28 days and were slaughtered (4 goat/group) at 5,7 and 9 h after butyrate infusion. Rumen fluid was analyzed for short chain fatty acids (SCFAs) concentration. Ruminal tissues were analyzed for morpho-histrometry and the expressions of genes associated with cell cycle and apoptosis. The results revealed that the ruminal butyrate concentration increased (P < 0.05) in B compared to group A. Morphometric analysis showed increased (P < 0.05) papillae size associated with higher number of cell layers in epithelial strata in B compared to A. Butyrate-induced papillae enlargement was coupled with enhanced mRNA expression levels (P < 0.05) of cyclin D1, CDK2, CDK4, and CDK6 (G0/G1 phase regulators) at 5 h, cyclin E1 (G1/S phase regulator) at 7 h and cyclin A and CDK1 (S phase regulators) at 9 h post-infusion compared to A group. In addition, the mRNA expression levels of apoptotic genes, i.e., caspase 3, caspase 9 and Bax at 5 h post-infusion were upregulated (P < 0.05) in group B compared to group A. The present study demonstrated that butyrate improved ruminal epithelial growth through concurrent and time-dependent changes in the expressions of genes involved in cell proliferation and apoptosis. It seems that the rate of proliferation was higher than the apoptosis which was reflected in epithelial growth.

Chenopodium quinoa is a crop with outstanding tolerance to saline soil, but long non-coding RNAs (LncRNAs) expression profile driven by salt stress in quinoa has rarely been observed yet. Based on the high-quality quinoa reference genome and high-throughput RNA sequencing (RNA-seq), genome-wide identification of LncRNAs was performed, and their dynamic response under salt stress was then investigated. In total, 153,751 high-confidence LncRNAs were discovered and dispersed intensively in chromosomes. Expression profile analysis demonstrated significant differences between LncRNAs and coding RNAs. Under salt stress conditions, 4,460 differentially expressed LncRNAs were discovered, of which only 54 were differentially expressed at all the stress time points. Besides, strongly significantly correlation was observed between salt-responsive LncRNAs and their closest neighboring genes (r = 0.346, p-value &lt; 2.2e-16). Furthermore, a weighted co-expression network was then constructed to infer the potential biological functions of LncRNAs. Seven modules were significantly correlated with salt treatments, resulting in 210 hub genes, including 22 transcription factors and 70 LncRNAs. These results indicated that LncRNAs might interact with transcription factors to respond to salinity stress. Gene ontology enrichment of the coding genes of these modules showed that they were highly related to regulating metabolic processes, biological regulation and response to stress. This study is the genome-wide analysis of the LncRNAs responding to salt stress in quinoa. The findings will provide a solid framework for further functional research of salt responsive LncRNAs, contributing to quinoa genetic improvement.

We examined whether surplus dietary selenium (Se) supply could alleviate high concentrate (HC) diet‐induced hepatic oxidative stress (OS) and inflammation. Eighteen young goats were distributed into three groups; were fed low (LC, concentrate: forage; 35: 65), high concentrate (HC, 65: 35), or Se‐supplemented HC (HCSe, 65: 35 + 0.5 mg Se kg−1 diet) diets for 10 weeks. Short chain fatty acids, OS markers and immunoinflammatory genes expressions were assessed through gas chromatograph, kits, and RT‐qPCR, respectively. Compared with LC, HC diet increased (p < .05) colonic and serum lipopolysaccharide (LPS) levels and induced hepatic oxidative injury by increasing (p < .05) malondialdehyde (MDA) levels and decreasing (p < .05) activities of glutathione peroxidase, superoxide dismutase, and catalase. HC diet altered hepatic mRNA expressions of toll‐like receptor‐4 (TLR‐4), cluster of differentiation‐14 (CD‐14), tumor necrosis factor‐α (TNF‐α), TNF receptor‐associated factor‐6 (TRAF‐6), nuclear factor kappa B (NF‐κB), interleukin‐1β (IL‐1β), IL‐10, IL‐13, LPS‐binding protein (LBP), serum amyloid A (SAA), α‐acid glycoprotein (AGP), and albumin (ALB). Conversely, extra‐Se supply lowered LPS and attenuated antioxidant status and inflammation in liver. In conclusion, HC diet induced oxidative lesions and TLR‐4 pathway‐mediated inflammation, whereas supranutritional Se alleviated oxidative and inflammatory lesions through TLR‐4 pathway regulation in goat liver.