Quail GHRL and LEAP2 gene cloning, polymorphism detection, phylogenetic analysis, tissue expression profiling and its association analysis with feed intake.

The GHRL, LEAP2, and GHSR system have recently been identified as important regulators of feed intake in mammals and chickens. However, the complete cloning of the quail GHRL (qGHRL) and quail LEAP2 (qLEAP2) genes, as well as their association with feed intake, remains unclear. This study cloned the entire qGHRL and qLEAP2 cDNA sequence in Chinese yellow quail (Coturnix japonica), including the 5' and 3' untranslated regions. Sanger sequencing analysis revealed no missense mutations in the coding region of qGHRL and qLEAP2. Subsequently, phylogenetic analysis and protein homology alignment were conducted on the qGHRL and qLEAP2 in major poultry species. The findings of this research indicated that the qGHRL and qLEAP2 sequences exhibit a high degree of similarity with those of chicken and turkey. Specifically, the N-terminal 6 amino acids of GHRL mature peptides and all the mature peptide sequence of LEAP2 exhibited consistent patterns across all species examined. The analysis of tissue gene expression profiles indicated that qGHRL was primarily expressed in the proventriculus and brain tissue, whereas qLEAP2 exhibited higher expression levels in the intestinal tissue, kidney, and liver tissue, differing slightly from previous studies conducted on chicken. It is necessary to investigate the significance of elevated expression of qGHRL in brain and qLEAP2 in kidney in the future. Further research has shown that the expression of qLEAP2 can quickly respond to changes in different energy states, whereas qGHRL does not exhibit the same capability. Overall, this study successfully cloned the complete cDNA sequences of qGHRL and qLEAP2, and conducted a comprehensive examination of their tissue expression profiles and gene expression levels in the main expressing organs across different energy states. Our current findings suggested that qLEAP2 is highly expressed in the liver, intestine, and kidney, and its expression level is regulated by feed intake.

Screening of reliable reference genes for the normalization of RT-qPCR in chicken oviduct tract.

Utilizing publicly available RNA-seq data to screen for ideal reference genes is more efficient and accurate than traditional methods. Previous studies have identified optimal reference genes in various chicken tissues, but none have specifically focused on the oviduct (including the infundibulum, magnum, isthmus, uterus, and vagina), which is crucial for egg production. Identifying stable reference genes in the oviduct is essential for improving research on gene expression levels. This study investigated genes with consistent expression patterns in the chicken oviduct, encompassing both individual oviduct tract tissues and the entire oviduct, by utilizing multiple RNA-seq datasets. The screening results revealed the discovery of 100 novel reference genes in each segment of oviduct tissues, primarily associated with cell cycle regulation and RNA binding. Moreover, the majority of housekeeping genes (HKGs) showed inconsistent expression levels across distinct samples, suggesting their lack of stability under varying conditions. The stability of the newly identified reference genes was assessed in comparison to previously validated stable reference genes in chicken oviduct and commonly utilized HKGs, employing traditional reference gene screening methods. HERPUD2, CSDE1, VPS35, PBRM1, LSM14A, and YWHAB were identified to be suitable novel reference gene for different parts of the oviduct. HERPUD2 and YWHAB were reliable for gene expression normalization throughout the oviduct tract. Furthermore, overexpression and interference assays in DF1 cells showed LSM14A and YWHAB play a crucial role in cell proliferation, highlighting the importance of these newly reference genes for further research. Overall, this study has expanded the options for reference genes in RT-qPCR experiments in different segments of the chicken oviduct and the entire oviduct.

Establishment of a Steatosis Model in LMH Cells, Chicken Embryo Hepatocytes, and Liver Tissues Based on a Mixture of Sodium Oleate and Palmitic Acid.

Research on hepatic steatosis in animal husbandry has been a prominent area of study. Developing an appropriate in vitro cellular steatosis model is crucial for comprehensively investigating the mechanisms involved in liver lipid deposition in poultry and for identifying potential interventions to address abnormalities in lipid metabolism. The research on the methods of in vitro liver steatosis in chickens, particularly the effects of different fat mixtures, is still lacking. In this study, LMH cells were utilized to investigate the effects of OA, SO, PA, SP, and their pairwise combinations on steatosis development, with the aim of identifying the optimal conditions for inducing steatosis. Analysis of triglyceride (TG) content in LMH cells revealed that OA and SP had limited efficacy in increasing TG content, while a combination of SO and PA in a 1:2 ratio exhibited the highest TG content. Moreover, Oil Red O staining results in LMH cells demonstrated that the combination treatment had a more pronounced induction effect compared to 0.375 mM SO. Additionally, RNA-seq analysis showed that 0.375 mM SO significantly influenced the expression of genes associated with fatty acid metabolism compared to the control group, whereas the combination of SO and PA led to an enrichment of key GO terms associated with programmed cell death. These findings suggest that varying conditions of cellular steatosis could lead to distinct disruptions in gene expression. The optimal conditions for inducing steatosis in LMH cells were also tested on chicken embryonic liver cells and embryos. TG detection and Oil Red O staining assays showed that the combination of SO and PA successfully induced steatosis. However, the gene expression pattern differed from that of LMH cells. This study lays the foundations for further investigations into avian hepatic steatosis.

Screening of reliable reference genes for the normalization of RT-qPCR in chicken liver tissues and LMH cells.

The liver plays a vital role in lipid synthesis and metabolism in poultry. To study the functional genes more effectively, it is essential to screen of reliable reference genes in the chicken liver, including females, males, embryos, as well as the Leghorn Male Hepatoma (LMH) cell line. Traditional reference gene screening involves selecting commonly used housekeeping genes (HKGs) for RT-qPCR experiments and using different algorithms to identify the most stable ones. However, this approach is limited in selecting the best reference gene from a small pool of HKGs. High-throughput sequencing technology may offer a solution to this limitation. This study aimed to identify the most consistently expressed genes by utilizing multiple published RNA-seq data of chicken liver and LMH cells. Subsequently, the stability of the newly identified reference genes was assessed in comparison to previously validated stable poultry liver expressed reference genes and the commonly employed HKGs using RT-qPCR. The findings indicated that there is a higher degree of similarity in stable expression genes between female and male liver (such as LSM14A and CDC40). In embryonic liver, the optimal new reference genes were SUDS3, TRIM33, and ERAL1. For LMH cells, the optimal new reference genes were ALDH9A1, UGGT1, and C21H1orf174. However, it is noteworthy that most HKGs did not exhibit stable expression across multiple samples, indicating potential instability under diverse conditions. Furthermore, RT-qPCR experiments proved that the stable expression genes identified from RNA-seq data outperformed commonly used HKGs and certain validated reference genes specific to poultry liver. Over all, this study successfully identified new stable reference genes in chicken liver and LMH cells using RNA-seq data, offering researchers a wider range of reference gene options for RT-qPCR in diverse situations.

Screening of reliable reference genes for the normalization of RT-qPCR in chicken gastrointestinal tract.

The application of reverse transcription quantitative real-time PCR technology for the production of gene tissue expression profiles is a widely employed approach in molecular biology research. It is imperative to ascertain internal reference genes that exhibit stable expression across diverse tissues to ensure the precision of tissue gene expression profiles. While there have been studies documenting the most suitable reference genes for various tissues in chickens, there is a dearth of research on the identification of reference genes in the gastrointestinal (GI) tract of chickens. This study utilized 4 different algorithms (Delta CT, BestKeeper, NormFinder, and Genorm) to assess the stability of 19 internal reference genes in various GI tract tissues, including individual GI tract tissues, the anterior and posterior GI tract, and the entire GI tissue. The RefFinder software was employed to comprehensively rank these genes. The research findings successfully identified the most appropriate internal reference genes for each type of GI tissue. Furthermore, TBP, DNAJC24, Polr2b, RPL13, andAp2m exhibited stable expression in the entire and posterior GI tract, whereas HMBS, TBP, Ap2m, GUSB, DNAJC24, and RPL13 demonstrated stable expression in the anterior GI tract. However, the internal reference genes commonly utilized, namely ß-Actin, 18s RNA, and ALB, exhibit poor stability and are not advised for future investigations concerning gene expression in the GI region. Consequently, MUC2 and CDX1, 2 genes that specifically express in the gut, were chosen for examination to ascertain the stability of the aforementioned internal reference genes in this particular study. In summary, this study presents a relatively stable set of internal reference genes that can be employed to enhance the precision of quantifying mRNA expression levels in functional genes within the chicken GI tract.

Chicken LEAP2 Level Substantially Changes with Feed Intake and May Be Regulated by CDX4 in Small Intestine.

Ghrelin O-acyltransferase (GOAT), ghrelin, and GHSR have been reported to play important roles that influence feed intake in mammals. LEAP2, an endogenous antagonist of GHSR, plays an important role in the regulation of feed intake. However, chicken ghrelin has also been reported to have an inhibitory effect on feed intake. The role of the GOAT-Ghrelin-GHSR-LEAP2 axis in chicken-feed intake remains unclear. Therefore, it is necessary to systematically evaluate the changes in the tissue expression levels of these genes under different energy states. In this study, broiler chicks in different energy states were subjected to starvation and feeding, and relevant gene expression levels were measured using quantitative real-time PCR. Different energy states significantly modulated the expression levels of LEAP2 and GHSR but did not significantly affect the expression levels of GOAT and ghrelin. A high expression level of LEAP2 was detected in the liver and the whole small intestine. Compared to the fed group, the fasted chicks showed significantly reduced LEAP2 expression levels in the liver and the small intestine; 2 h after being refed, the LEAP2 expression of the fasted chicks returned to the level of the fed group. Transcription factor prediction and results of a dual luciferase assay indicated that the transcription factor CDX4 binds to the LEAP2 promoter region and positively regulates its expression. High expression levels of GHSR were detected in the hypothalamus and pituitary. Moreover, we detected GHSR highly expressed in the jejunum-this finding has not been previously reported. Thus, GHSR may regulate intestinal motility, and this aspect needs further investigation. In conclusion, this study revealed the function of chicken LEAP2 as a potential feed-intake regulator and identified the potential mechanism governing its intestine-specific expression. Our study lays the foundations for future studies on avian feed-intake regulation.