Leucine-Mediated SLC7A5 Promotes Milk Protein and Milk Fat Synthesis through mTOR Signaling Pathway in Goat Mammary Epithelial Cells.

The SLC7A5 gene encodes a Na+ and pH-independent transporter protein that regulates cell growth by regulating the uptake of AA. This study, utilizing RNA-seq, aimed to explore the effect of SLC7A5 on the synthesis of milk proteins and fats in goat mammary epithelial cells (GMECs) through gene interference and overexpression techniques. The results demonstrated that the overexpression of SLC7A5 resulted in a significant increase in the expression of CSN1S1, SCD, CEBPB, ACACA, αS1-casein, p-S6K, and p-S6. The levels of p-S6K and p-S6 gradually increased as the AA/Leu stimulation time lengthened. The overexpression of SLC7A5 rescued the role of Torin1 in GMECs. In conclusion, SLC7A5 plays a crucial role in promoting the synthesis of milk proteins and milk fats through the mTOR signaling pathway in GMECs, providing a theoretical foundation for improving the quality of goat milk.

Novel mechanism of Clostridium butyricum alleviated coprophagy prevention-induced intestinal inflammation in rabbit.

As bacteria synthesize nutrients primarily in the cecum, coprophagy is indispensable for supplying rabbits with essential nutrients. Recent research has demonstrated its pivotal role in maintaining intestinal microbiota homeostasis and immune regulation in rabbits, although the specific mechanism remains unknown. Here, we used coprophagy prevention (CP) to investigate the effects of coprophagy on the cecum homeostasis and microbiota in New Zealand white rabbits. Furthermore, whether supplementation of Clostridium butyricum (C. butyricum) may alleviate the cecum inflammation and apoptosis caused by CP was also explored. Four groups were randomly assigned: control (Con), sham-coprophagy prevention (SCP), coprophagy prevention (CP), and CP and C. butyricum addition (CPCB). Compared to Con and SCP, CP augmented cecum inflammation and apoptosis, as well as bacterial adhesion to the cecal epithelial mucosa, while decreasing the expression of tight junction proteins (ZO-1, occluding, and claudin-1). The relative abundance of short-chain fatty acids (SCFAs)-producing bacteria was significantly decreased in the CP group. Inversely, there was an increase in the Firmicutes/Bacteroidetes ratio and the relative abundance of Christensenellaceae_R-7_group. Additionally, CP increased the levels of Flagellin, IFN-γ, TNF-a, and IL-1ß in cecum contents and promoted the expression of TLR5/MyD88/NF-κB pathway in cecum tissues. However, the CPCB group showed significant improvements in all parameters compared to the CP group. Dietary C. butyricum supplementation significantly increased the production of SCFAs, particularly butyric acid, triggering anti-inflammatory, tissue repairing, and barrier-protective responses. Notably, CPCB effectively mitigated CP-induced apoptosis and inflammation. In summary, CP disrupts the cecum epithelial barrier and induces inflammation in New Zealand white rabbits, but these effects can be alleviated by C. butyricum supplementation. This process appears to be largely associated with the TLR5/MyD88/NF-κB signaling pathway.

Impact of coprophagy prevention on the growth performance, serum biochemistry, and intestinal microbiome of rabbits.

BACKGROUND: Coprophagy plays a vital role in maintaining growth and development in many small herbivores. Here, we constructed a coprophagy model by dividing rabbits into three groups, namely, control group (CON), sham-coprophagy prevention group (SCP), and coprophagy prevention group (CP), to explore the effects of coprophagy prevention on growth performance and cecal microecology in rabbits. RESULTS: Results showed that CP treatment decreased the feed utilization and growth performance of rabbits. Serum total cholesterol and total triglyceride in the CP group were remarkably lower than those in the other two groups. Furthermore, CP treatment destroyed cecum villi and reduced the content of short-chain fatty acids (SCFAs) in cecum contents. Gut microbiota profiling showed significant differences in the phylum and genus composition of cecal microorganisms among the three groups. At the genus level, the abundance of Oscillospira and Ruminococcus decreased significantly in the CP group. Enrichment analysis of metabolic pathways showed a significantly up-regulated differential metabolic pathway (PWY-7315, dTDP-N-acetylthomosamine biosynthesis) in the CP group compared with that in the CON group. Correlation analysis showed that the serum biochemical parameters were positively correlated with the abundance of Oscillospira, Sutterella, and Butyricimonas but negatively correlated with the abundance of Oxalobacte and Desulfovibrio. Meanwhile, the abundance of Butyricimonas and Parabacteroidesde was positively correlated with the concentration of butyric acid in the cecum. CONCLUSIONS: In summary, coprophagy prevention had negative effects on serum biochemistry and gut microbiota, ultimately decreasing the growth performance of rabbits. The findings provide evidence for further revealing the biological significance of coprophagy in small herbivorous mammals.

Microbiome-Metabolome Analysis of the Immune Microenvironment of the Cecal Contents, Soft Feces, and Hard Feces of Hyplus Rabbits.

The intestinal microbiota and its metabolites play vital roles in host growth, development, and immune regulation. This study analyzed the microbial community distribution and the cytokine and short-chain fatty acid (SCFA) content of cecal contents (Con group), soft feces (SF group), and hard feces (HF group) of 60-day-old Hyplus rabbits and verified the effect of soft feces on the cecal immune microenvironment by coprophagy prevention (CP). The results showed that there were significant differences in the levels of phylum and genus composition, cytokines, and SCFAs among the Con group, SF group, and HF group. The correlation analysis of cytokines and SCFAs with differential microbial communities showed that Muribaculaceae, Ruminococcaceae_UCG-014, Ruminococcaceae_NK4A214_group, and Christensenellaceae_R-7_Group are closely related to cytokines and SCFAs. After CP treatment, the contents of propionic acid, butyric acid, IL-4, and IL-10 in cecum decreased significantly, whereas TNF-α and IL-1ß increased significantly. Moreover, the inhibition of coprophagy led to the downregulation of the expression levels of tight junction proteins (Claudin-1, Occludin, and ZO-1) related to intestinal inflammation and intestinal barrier function, and the ring-like structure of ZO-1 was disrupted. In conclusion, coprophagy can not only help rabbits obtain more probiotics and SCFAs but also play an essential role in improving the immune microenvironment of cecum.

Selection and validation of reference genes for the normalization of quantitative real-time PCR in different muscle tissues of rabbits.

BACKGROUND: In molecular biology studies, the selection of optimal reference genes is of vital importance for accurately quantifying gene expression. The purpose of the present study was to screen the most stable reference genes in different muscle tissues of New Zealand white rabbits and Yufeng yellow rabbits. METHODS AND RESULTS: Results indicated that the most stable reference genes in the muscle tissues of New Zealand white rabbits were HPRT1, ACTB and PPIC, while HPRT1, PPIC, and RPL13A were the most stable reference genes in muscle tissues of Yufeng yellow rabbits. However, in the longissimus dorsi muscle and the abdominal wall muscle of both varieties, the most stable reference genes were HPRT1, RPL13A, and SDHA. In the quadriceps femoris muscle, the most stable reference genes were ACTB, HPRT1, and SDHA. Furthermore, the relative abundance of MYOG, MYH3 and MSTN was used to confirm the suitability and reliability of the selected most stable reference genes and the most unstable reference gene. Results revealed the same expression patterns of these myogenic genes when normalized according to the most stable genes, while normalization against the unstable reference gene altered the observed expression patterns. CONCLUSIONS: Taken together, our results demonstrated that the most stable reference genes varied among different muscle tissues and different breeds of rabbits. However, HPRT1, PPIC and SDHA presented high stability among all examined reference genes; thus, the combined analysis of HPRT1/ PPIC/ SDHA gene provides the best reference for RT-qPCR in muscle tissues of New Zealand white rabbits and Yufeng yellow rabbits, while HPRT1 is a better choice than other reference genes when using a single reference gene to assess target gene expression. Our results provide basic data for better measuring target gene expression profiles in muscle tissues of rabbits.

Screening and stability analysis of reference genes in fasting caecotrophy model in rabbits.

BACKGROUND: The selection and validation of stably expressed reference genes is key for accurately quantifying the mRNA abundance of genes under different treatments. In the rabbit model of fasting caecotrophy, reports about the selection of stable reference genes are not available. METHODS AND RESULTS: This study aims to screen suitable reference genes in different tissues (including uterus, cecum, and liver) of rabbits between control and fasting caecotrophy groups. RT-qPCR was used to analyze the expression levels of eight commonly used reference genes (including GAPDH, 18S rRNA, B2M, CYP, HPRT1, ß-actin, H2afz, Ywhaz), and RefFinder (including geNorm, NormFinder, and BestKeeper) was used to analyze the expression stability of these reference genes. Our results showed that the most stable reference genes were different in different tissues and treatments. In the control and fasting caecotrophy groups, CYP, GAPDH and HPRT1 were proven to be the top stable reference genes in the uterus, cecum, and liver tissues, respectively. GAPDH and Ywhaz were proven to be the top two stable reference genes among uterus, cecum, and liver in both control and fasting caecotrophy groups. CONCLUSIONS: Our results indicated that the combined analysis of three or more reference genes (GAPDH, HPRT1, and Ywhaz) are recommended to be used for RT-qPCR normalization in the rabbit model of fasting caecotrophy, and that GAPDH is a better choice than the other reference genes for normalizing the relative expression of target genes in different tissues of fasting caecotrophy rabbits.