MiRNA-21-5p induces chicken hepatic lipogenesis by targeting NFIB and KLF3 to suppress the PI3K/AKT signaling pathway.

The liver plays a critical role in metabolic activity and is the body's first immune barrier, and maintaining liver health is particularly important for poultry production. MicroRNAs (miRNAs) are involved in a wide range of biological activities due to their capacity as posttranscriptional regulatory elements. A growing body of research indicates that miR-21-5p plays a vital role as a modulator of liver metabolism in various species. However, the effect of miR-21-5p on the chicken liver is unclear. In the current study, we discovered that the fatty liver had high levels of miR-21-5p. Then the qPCR, Western blot, flow cytometry, enzyme-linked immunosorbent assay, dual-luciferase, and immunofluorescence assays were, respectively, used to determine the impact of miR-21-5p in the chicken liver, and it turned out that miR-21-5p enhanced lipogenesis, oxidative stress, and inflammatory responses, which ultimately induced hepatocyte apoptosis. Mechanically, we verified that miR-21-5p can directly target nuclear factor I B (NFIB) and kruppel-like factor 3 (KLF3). Furthermore, our experiments revealed that the suppression of NFIB promoted apoptosis and inflammation, and the KLF3 inhibitor accelerated lipogenesis and enhanced oxidative stress. Furthermore, the cotransfection results suggest that the PI3K/AKT pathway is also involved in the process of miRNA-21-5p-mediate liver metabolism regulation. In summary, our study demonstrated that miRNA-21-5p plays a role in hepatocyte lipogenesis, oxidative stress, inflammation, and apoptosis, via targeting NFIB and KLF3 to suppress the PI3K/AKT signal pathway in chicken.

miR-21-5p is a typical noncoding RNA that could inhibit messenger RNA expression by targeting the 3ʹ-untranslated region to participate in fatty liver-related disease formation and progression. We demonstrated that miRNA-21-5p plays a role in hepatocyte lipogenesis, oxidative stress, inflammation, and apoptosis, via targeting nuclear factor I B and kruppel-like factor 3 to suppress the PI3K/AKT signal pathway in chicken. This research established the regulatory network mechanisms of miR-21-5p in chicken hepatic lipogenesis and fatty liver syndrome.

Thiram exposure induces tibial dyschondroplasia in broilers via the regulation effect of circ_003084/miR-130c-5p/BMPR1A crosstalk on chondrocyte proliferation and differentiation.

Thiram, an agricultural insecticide, has been demonstrated to induce tibial dyschondroplasia (TD) in avian species. Circular RNA (circRNAs), a novel class of functional biological macromolecules characterized by their distinct circular structure, play crucial roles in various biological processes and diseases. Nevertheless, the precise regulatory mechanism underlying non-coding RNA involvement in thiram-induced broiler tibial chondrodysplasia remains elusive. In this study, we established a broiler model of thiram exposure for 10 days to assess TD and obtain a ceRNA network by RNA sequencing. By analyzing the differentially expressed circRNAs network, we id entify that circ_003084 was significantly upregulated in TD cartilage. Elevated circ_003084 inhibited TD chondrocytes proliferation and differentiation in vitro but promote apoptosis. Mechanistically, circ_003084 competitively binds to miR-130c-5p and prevents miR-130c-5p to decrease the level of BMPR1A, which upregulates the expression of apoptosis genes Caspase 3, Caspase 9, Bax and Bcl2, and finally facilitates cell apoptosis. Taken together, these findings imply that circ_003084/miR-130c-5p/BMPR1A interaction regulated TD chicken chondrocyte proliferation, apoptosis, and differentiation. This is the first work to reveal the mechanism of regulation of circRNA-related ceRNA on thiram-induced TD, offering a key reference for environmental toxicology.

Lipid Deposition and Progesterone Synthesis Are Increased by miR-181b-5p through RAP1B/ERK1/2 Pathway in Chicken Granulosa Cells.

Steroid hormones secreted by granulosa cells are essential for maintaining normal development of chicken follicles. Our previous sequencing data indicated that miR-181b-5p and RAS-related protein 1B (RAP1B) appeared to function in chicken granulosa cells, which was further explored in this study. The results suggested that miR-181b-5p facilitated the aggregation of lipid droplets and the synthesis of progesterone. In contrast, RAP1B astricted lipid deposition and progesterone secretion. Cotransfection of the RAP1B overexpression vector with miR-181b-5p mimic eliminated the promoting effect of miR-181b-5p. Dual-luciferase reporter assay confirmed that miR-181b-5p bound directly to the 3' untranslated region (3' UTR) of RAP1B. We also found that miR-181b-5p and RAP1B reduced and enhanced the phosphorylation levels of extracellular signal-regulated kinases 1 and 2 (ERK1/2), respectively. The application of ERK1/2 activators and inhibitors demonstrated that ERK1/2 is a negative regulator of lipid deposition and progesterone synthesis. In conclusion, we revealed that miR-181b-5p accelerated lipid deposition and progesterone synthesis through the RAP1B/ERK1/2 pathway in chicken granulosa cells. miR-181b-5p and RAP1B may serve as new biomarkers in breeding to improve chicken reproductive performance and prevent ovary-related diseases.

RNA sequencing reveals lncRNA-mediated non-mendelian inheritance of feather growth change in chickens.

BACKGROUND: Long non-coding RNAs (lncRNAs) play an essential role in biological processes. However, the expression patterns of lncRNAs that regulate the non-Mendelian inheritance feather phenotypes remain unknown. OBJECTIVE: This study aimed to compare the expression profiles of lncRNAs in the follicles of the late-feathering cocks (LC) and late-feathering hens (LH) that followed genetic rules and the early-feathering hen (EH) and early-feathering cock (EC) that did not conform to the genetic laws. METHODS: We performed RNA sequencing and investigated the differentially expressed lncRNAs (DElncRNAs) between the early- and late-feathering chickens, which function by cis-acting or participate in the competing endogenous RNA (ceRNA) network. RESULTS: A total of 53 upregulated and 43 downregulated lncRNAs were identified in EC vs. LC, and 58 upregulated and 109 downregulated lncRNAs were identified in EH vs. LH. The target mRNAs regulated by lncRNAs in cis were enriched in the pentose phosphate pathway, TGF-ß signaling pathway and Jak-STAT signaling pathway in EC vs. LC and were associated with the TGF-ß signaling pathway, Wnt signaling pathway, p53 signaling pathway and Jak-STAT signaling pathway in EH vs. LH. In addition, the lncRNA-mediated ceRNA regulatory pathways of hair follicle formation were mainly enriched in the TGF-ß signaling pathway, Wnt signaling pathway, melanogenesis, and calcium signaling pathways. The levels of ENSGALG00000047626 were significantly higher in the late-feathering chickens than in the early-feathering chickens, which regulated the expression of SSTR2 by gga-miR-1649-5p. CONCLUSION: This study provides a novel molecular mechanism of lncRNA's response to the feather rate that does not conform to the genetic laws in chickens.

New Insights into the Hourly Manure Coverage Proportion on the Manure Belt in a Typical Layer House for Accurate Ammonia Emission Modeling.

The main advantage of having livestock, for example, the laying hens, in a controlled environment is that the optimum growth conditions can be achieved with accuracy. The indoor air temperature, humidity, gases concentration, etc., would significantly affect the animal performance, thus should be maintained within an acceptable range. In order to achieve the goals of precision poultry farming, various models have been developed by researchers all over the world to estimate the hourly indoor environmental parameters so as to provide decision suggestions. However, a key parameter of hourly manure area in the poultry house was missing in the literature to predict the ammonia emission using the recently developed mechanistic model. Therefore, in order to fill the gap of the understanding of hourly manure coverage proportion and area on the manure belt, experimental measurements were performed in the present study using laying hens from 10 weeks age to 30 weeks age. For each test, six polypropylene (pp) plates were applied to collect the manure dropped by the birds every hour, and photographs of the plates were taken at the same time using a pre-fixed camera. Binary images were then produced based on the color pictures to determine the object coverage proportion. It was demonstrated that for laying hens of stocking density around 14 birds/m2, the manure coverage proportion at the 24th hour after the most recent manure removal was about 60%, while the value was approximately 82% at the 48th hour. Meanwhile, for laying hens at different ages, the hourly increment of manure coverage proportion showed a similar pattern with four distinct stages within 48 h. The statistical analyses demonstrated no significant correlation between the hourly increment of manure weight and the hourly increment of manure coverage proportion. Finally, prediction models for estimating the hourly manure coverage proportion on the manure belt in typical laying hen houses were provided.

Transcriptome sequencing reveals genes involved in cadmium-triggered oxidative stress in the chicken heart.

As a ubiquitous heavy metal, cadmium (Cd) is highly toxic to various organs. However, the effects and molecular mechanism of Cd toxicity in the chicken heart remain largely unknown. The goal of our study was to investigate the cardiac injury in chickens' exposure to Cd. We detected the levels of oxidative stress-related molecules in the Cd-induced chicken heart, and assessed the histopathological changes by hematoxylin and eosin staining. RNA sequencing was performed to identify differentially expressed mRNAs between the Cd-induced group and control group. The expression of candidate genes involved in oxidative stress was certified by quantitative reverse transcription PCR. Our results showed that the expression of glutathione, peroxidase, and superoxide dismutase was significantly decreased and malondialdehyde was increased in the heart of chickens by Cd induction. The disorderly arranged cardiomyocytes, swelled and enlarged cells, partial cardiomyocyte necrosis, blurred morphological structure, and notable inflammatory cell infiltration were observed in the Cd-induced chicken heart. RNA sequencing identified 23 upregulated and 11 downregulated mRNAs in the heart tissues of the chicken in the Cd-induced group, and functional pathways indicated that they were associated with oxidative stress. Moreover, CREM, DUSP8, and ITGA11 expressions were significantly reduced, whereas LAMA1 expression was induced in heart tissue of chickens by Cd treatment. Overall, our findings revealed that oxidative stress and pathological changes in the chicken heart could be triggered by Cd. The mRNA transcriptional profiles identified differentially expressed genes in the chicken heart by Cd induction, revealing oxidative stress-related key genes and enhancing our understanding of Cd toxicity in the chicken heart.

Long Non-coding RNA Expression Profile in Broiler Liver with Cadmium-Induced Oxidative Damage.

Cadmium pollution is serious heavy metal pollution in environmental pollution and impacts on livestock productivity. However, the effect and mechanisms of cadmium toxicity on the broiler remain unclear. This study aimed to explore the liver oxidative damage and reveal the related long non-coding RNA (lncRNA) expression patterns in the broiler liver with cadmium exposure. The broilers were fed with diets containing CdCl2 and detected the oxidative stress indexes in the liver tissues. Transcriptome sequencing of broiler liver was performed to identify cadmium exposure-related differentially expressed lncRNAs (DElncRNAs). The functions and pathways of DElncRNAs were analyzed by GO and KEGG. The sequencing results were verified by the quantitative real-time polymerase chain reaction. Cadmium exposure induced tissue structure disorder, focal hemorrhage, and irregular hepatocytes in the broiler liver, and significantly decreased GSH level and enzyme activities, and increased MDA expression in the liver. A total of 74 DElncRNAs were obtained in cadmium group compared with the control group, which were enriched in the GO terms, including intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator, branched-chain amino acid biosynthetic process. The enriched KEGG pathways, including lysine biosynthesis, valine, leucine and isoleucine biosynthesis, and pantothenate and CoA biosynthesis, were related to oxidative stress. PCR analysis indicated that the changes in ENSGALG00000053559, ENSGALG00000053926, and ENSGALG00000054404 expression were consistent with sequencing. Our results provide novel lncRNAs involved in oxidative stress in the broiler liver with cadmium exposure.

Peroxisome proliferator-activated receptor-coactivator 1-beta (PGC-1ß) modulates the expression of genes involved in adipogenesis during preadipocyte differentiation in chicken.

To study the influence of the PGC-1ß gene on chicken adipocyte proliferation and differentiation, we constructed RNA interference (RNAi) vectors that target the PGC-1ß gene and transfected these vectors into adipocytes. Oil Red O staining and a CCK-8 cell kit were used to determine cell triglyceride accumulation status and cell proliferation after transfection, respectively. The mRNA abundances of PGC-1ß and adipocyte-differentiation-related genes (PPARγ, C/EBPα, SREBP-1c, FAS, and A-FABP) were detected by real-time PCR. The results showed that the mRNA and protein abundances of PGC-1ß in PGC-1ß-shRNA transfected adipocytes were significantly lower than those in the control. Interference decreased cell differentiation, but did not depress the cell proliferation. PGC-1ß interference impeded the triglyceride accumulation, the mRNA expression levels of nuclear receptors PPARγ and SREBP-1c, and fatty acid synthetase (FAS), and both proteins PPARγ and SREBP-1c, and the fatty acids transporting protein A-FABP. Generally, PGC-1ß modulated the cell differentiation and triglyceride accumulation in chicken adipocytes.

Genotypes of IFIH1 and IFIT5 in seven chicken breeds indicated artificial selection for commercial traits influenced antiviral genes.

Innate immunity is the first line against the invasion of pathogenic microorganisms. Previous reports only demonstrated production traits of commercial importance were often negatively correlated with innate disease resistance. However, whether different purpose of artificial selection influences innate immunity have not been understood. In this study, we cloned exon1, exon6 of IFIH1 and exon2 of IFIT5 by molecular biology techniques in seven different chicken breeds to detect the potential effect of artificial selection for commercial traits on disease resistance for the first time. In total, 8 single nucleotide polymorphisms(SNPs) of IFIH1 gene exon1 and exon6, 19 SNPs of IFIT5 gene exon2 were detected. We found all native chicken breeds had a relatively close relationship to broiler breeds but a remote relationship to layer breed. A great difference between CB and LLH with different selected purpose were observed. The allele frequencies of these two positive antiviral genes were associated with different purpose of artificial selection. Our experiment constituted the foundation for the interaction between commercial traits and immune trait.