Search

Abstract Background Circular RNAs (circRNAs) are stable, covalently closed non-coding RNAs formed by reverse splicing of precursor mRNA. They play critical roles in various biological processes, including insulin secretion and metabolism. However, their function in avian skeletal muscle's response to insulin remains poorly understood. This study aimed to comprehensively identify insulin-responsive circRNAs and explore their temporal and breed-specific regulation in poultry. Results Using strand-specific RNA sequencing (ssRNA-Seq) on the pectoralis muscles of both Arbor Acres (AA) broilers and Silky fowls following insulin administration (5 IU/kg.BW, PBS as control). We identified 2,027 muscle circRNAs. Insulin-responsive circRNAs were detected in Silky fowls (29) and broilers (45) at 120 min, and in broilers (20) at 15 min post-injection. These circRNAs are enriched in processes such as exocrine pancreas development, response to exogenous stimuli, and regulation of intracellular signal transduction, likely mediated through a circRNA-miRNA network. Fewer insulin-responsive circRNAs were shared between time points in broilers (1) or between breeds (3) at 120 min. We further characterized a conserved insulin-responsive circRNA (circINSR), formed by exon 2 of the Insulin Receptor (INSR). The circINSR showed a similar spatiotemporal expression pattern to INSR, but exhibited distinct changes post-insulin administration. In broilers, INSR expression was dynamically modulated, while circINSR was downregulated only at 15 min (P

FK506-binding protein 5 (FKBP5) is a negative regulator of the glucocorticoid response and may play an important role in regulating metabolic homeostasis in birds. However, limited information is available regarding its role in avian species. This study aimed to clarify the spatiotemporal characteristics of chicken FKBP5 and investigate the effects of exogenous stimuli on its expression. Real-time quantitative PCR (RT-qPCR) was utilized to analyze the spatiotemporal expression patterns of FKBP5 in chickens. Additionally, the impact of exogenous stimuli, including fasting, energy restriction, and insulin/pyruvate/glucose injections, on FKBP5 expression in the pectoral muscle was investigated. The results showed that FKBP5 is broadly expressed in various bird tissues, with dominant expression in insulin-sensitive tissues. The FKBP5 levels in birds are dramatically modulated during development in insulin-sensitive tissues, with the highest expression observed in striated muscle and liver at embryonic day 19 (E19) (P < 0.01). The basal level of FKBP5 in the pectoral muscle of broilers is higher than that in Silky chickens. Insulin administration leads to a rapid drop in blood glucose levels in both breeds, with a slower recovery in AA broilers (P < 0.01). However, FKBP5 expression in the pectoral muscle initially shows a slight drop, followed by a sharp increase over time after insulin administration (P < 0.01). Additionally, breed heterogeneity in FKBP5 expression was observed in the pectoral muscle upon insulin stimulation, with broilers showing stronger insulin sensitivity compared to Silky chickens. A 24-hour fasting period downregulated blood glucose levels (P < 0.01) and FKBP5 expression in the pectoral muscle of AA broilers (P < 0.01). A 30% energy restriction over three weeks slightly reduced blood glucose levels (P = 0.075) and significantly decreased FKBP5 levels in the pectoralis muscle of broilers. In addition, pyruvate increased blood glucose and pectoralis FKBP5 transcription levels at 60 min (P < 0.01), whereas glucose increased blood glucose levels but had no effect on FKBP5 expression. Overall analysis showed a negative correlation between FKBP5 expression in the pectoral muscle and blood glucose levels (ρ = −0.405, P < 0.001). In conclusion, FKBP5 is a key player in the metabolic regulation of birds, with its expression being highly dynamic and tissue-specific. Insulin regulates FKBP5 expression in pectoral muscles in a time-dependent manner, while pyruvate increases FKBP5 levels and fasting/energy restriction reduces FKBP5 mRNA expression in the pectoral muscles.

Birds' glycolipid metabolism has garnered considerable attention due to their fasting blood glucose levels being nearly twice those of mammals. While skeletal muscle is the primary insulin-sensitive tissue in mammals, the effects of insulin on chicken skeletal muscle remain unclear. In this study, the insulin-responsive metabolites were identified in broiler's pectoralis muscle (after 16 h of fasting) using widely targeted metabolomics. Glycolipid concentrations were measured using kits, and the expression of key genes involved in glucose metabolism was assessed via quantitative real-time PCR (qRT-PCR). The insulin tolerance test, performed by injecting 5 IU/kg body weight of insulin, demonstrated a rapid drop in blood glucose levels from 0 to 15 min, with a consistent reduction observed at 120 min (P < 0.01). Insulin did not alter glucose and glycogen content in chicken pectoralis; however, low-density lipoprotein (LDL, P < 0.05) levels were upregulated in the early phase (15 min). With an extended insulin duration (120 min), pectoralis glucose content increased (P < 0.05), accompanied by a reduction in TG levels (P < 0.05). Metabolomic analysis revealed that insulin promotes the downregulation of 63 out of 71 metabolites at 15 min and the upregulation of 101 out of 134 metabolites at 120 min, mainly associated with lysine degradation and thyroid hormone signaling pathways, respectively. 7 metabolites were dynamically modulated in the same manner over time (2 up-up and 5 down-down). Early insulin inhibited glycolysis, evidenced by the reduction in phosphoenolpyruvate levels and hexokinase 2 (HK2) expression; however, insulin promoted glucose uptake through the activation of glucose transporter 4 (GLUT4) and enhanced glycolysis, accompanied by elevated fatty acid metabolism at the later phase.In conclusion, insulin dynamically regulates the metabolomics of the pectoralis muscle over time. Initially, chicken muscle tissues downregulate metabolic activities to accommodate the new signaling state, followed by significant upregulation to meet heightened metabolic demands. Extended insulin monitoring promotes glucose uptake and glycolysis, alongside enhanced fatty acid metabolism. This research provides insights into the potential mechanisms of insulin action in chicken muscles.

ABSTRACT: Glucose Transporter 4 (GLUT4) is a crucial protein facilitating glucose uptake and metabolism across cell membranes in mammals. However, information on GLUT4 in birds has historically been limited. In this study, we investigated the dynamic expression profile of chicken GLUT4 using real-time quantitative PCR (RT-qPCR) and examined its potential effects and mechanisms via GLUT4 overexpression and RNA sequencing (RNA-seq) in chicken primary skeletal muscle satellite cells (CP-SMSCs). Our results demonstrated that chicken GLUT4 is differentially expressed across tissues, with predominant expression in skeletal muscles, and across developmental stages of CP-SMSCs, with notable upregulation during the phases of cell proliferation and early differentiation. Notably, 0.1 μM insulin for 60 min significantly elevated the expression of GLUT4 in CP-SMSCs (P < 0.05). GLUT4 overexpression in CP-SMSCs promoted cell proliferation, as evidenced by Cell Counting Kit-8 (CCK-8) (P < 0.05) and 5-Ethynyl-2′-Deoxyuridine (EDU) assays (P < 0.05), and enhanced glucose consumption following 0.1 μM insulin treatment (P < 0.05). However, it inhibited glucose consumption 12 h after the addition of 5 g/L glucose (P < 0.05). After overexpressing GLUT4, we identified 302 differentially expressed genes (DEGs) in CP-SMSCs, with 134 upregulated and 168 downregulated. These DEGs are primarily enriched in pathways such as oxidative phosphorylation, ribosome, cardiac muscle contraction, ATP metabolic processes, and mitochondrial protein complexes. Specifically, in the enriched oxidative phosphorylation pathway, the upregulated DEGs (12) encode mitochondrial proteins, while the downregulated DEGs (6) are nuclear genome-derived. The ribosomal pathway is predominantly inhibited, accompanying with the downregulation of the translocase of outer mitochondrial membrane 7 (TOMM7)/translocase of inner mitochondrial membrane 8 (TIMM8A) complex responsible for mitochondrial protein transport, and a reduction in 28S (LOC121106978) and 18S (LOC112533601) ribosomal rRNAs. In conclusion, chicken GLUT4 is dynamically modulated during development and acts as an insulin responder that significantly regulates cellular glucose uptake and cell proliferation. This regulation occurs mainly through enhancing the mitochondrial oxidative phosphorylation and inhibiting ribosomal pathway.

ABSTRACT: Animal aggression is one of the most conserved behaviors. Excessive and inappropriate aggression was a serious social concern across species. After long-term selection under strict stress conditions, Henan gamecock serves as a good model for studying aggressive behavior. In this research, we constructed a Henan game chicken backcross population containing 25% Rhode Island Red (RIR), and conducted brain transcriptomics and serum metabolomics analyses on Henan gamecock (HGR) through its comparison with its female encounters (HGH) and the male backcross birds (BGR). The study revealed that seven differential metabolites in serum and 172 differentially expressed genes in the brain were commonly shared in both HGR vs. HGH and HGR vs. BGR comparisons. They exhibited the same patterns of modulation in Henan gamecocks, following either HGH < HGR > BGR or HGH > HGR < BGR style. Therein, some neurological genes involving in serotonergic and dopaminergic signaling were upregulated, while the levels of many genes related with neuro-immune function were decreased in Henan gamecock. In addition, many unknown genes specifically or highly expressed in the brain of the Henan gamecock were identified. These genes are potentially key candidates for enhancing the bird's aggression. Multi-omics joint analysis revealed that tyrosine metabolism and neuroactive ligand-receptor interaction were commonly affected. Overall, our results propose that the aggressiveness of Henan gamecocks can be heightened by the activation of the serotonergic-dopaminergic metabolic process in the brain, which concurrently impairs the neuroimmune system. Further research is needed to identify the function of these unknown genes on the bird's aggressive behavior.

The modern livestock industry faces significant challenges due to the extension of production cycles and a growing shortage of feed resources. Low-protein (LP) diets offer potential environmental advantages whereas damage intestinal integrity and increase pro-inflammatory cytokines. Bile acids (BAs), metabolic products of cholesterol in the liver, possess strong antioxidative and immune properties. This study evaluated the effects of dietary porcine BA supplementation at different protein levels on the antioxidant status, immune function, and gut microbiota of late-laying hens. A 2 × 2 factorial design was employed with 198 Hy-Line Brown laying hens (62 weeks old), randomly divided into one of four treatment groups with 8 replicates for each treatment. Diets included normal (16.42%) or low-protein (15.35%) levels, with porcine BAs supplemented at 0 or 120 mg/kg (62–69 weeks) and 0 or 200 mg/kg (70–75 weeks) over 14 weeks. Dietary protein levels and bile acids had no effect on organ development in laying hens. Low-protein diets increased serum anti-inflammatory cytokines IL-4 and IL-10 but elevated pro-inflammatory cytokines IL-1β (p < 0.05), corroborated by higher IL-10 gene expression in the ileum mucosa (p < 0.05). The supplementation of bile acids improved immune function by increasing serum IL-4 and TGF-β while decreasing IL-6 levels (p < 0.05). Moreover, bile acids treatments upregulated IL-10, TGF-β and ZO-1 gene expression in the ileum mucosa (p < 0.05) and alleviated oxidative stress by enhancing serum GSH-Px activity (p < 0.05). Additionally, both low-protein diets and bile acids enriched beneficial cecal bacteria, including Hungatella and Blautia, primarily linked to immune function. These findings demonstrate that porcine bile acids administration improve antioxidant capacity, immune function and gut microbiota dysbiosis. These beneficial effects were associated with Hungatella and Blautia levels, which may be responsible for the porcine bile acids efficacy in poultry nutrition.

IntroductionThe study aimed to investigate the potential effects of varying wheat levels in broiler diets on growth performance, intestinal barrier, and cecal microbiota.MethodsDay-old male broilers were fed the same diet until 10 d of age. Then they were randomly assigned to 1) the low-level wheat group, where inclusion of 15.0% and 25.0% wheat in the grower and finisher diet, respectively, 2) the medium-level wheat group with 30.0% and 40.0% of wheat in the grower and finisher periods; and 3) the high-level wheat dietary group, in which the grower and finisher diets contained 55.77% and 62.38% of wheat, respectively.ResultsDietary treatments unaffected the body weight at 39 d, whereas incorporating high wheat in diets significantly increased the feed intake and reduced the feed conversion ratio from 10 to 39 d (p

Multiply adverse effects including declines in production performance and excessive fat deposition were noticed with the extension of the laying cycle in hens, which are pertinent to animal welfare and human food safety. This study aimed to investigate the effect of dietary supplementation of bile acids (BAs) on production performance and lipid metabolism in late-phase laying hens. A total of 144 70-week-old hens were distributed into three treatments with eight replicates per treatment, including the basal diet with 0 (Ctrl), 95.01 (Low-BA), and 189.99 mg/kg (High-BA) of porcine BAs, respectively. The test period was from 70 to 75 weeks. The supplementation of BAs did not significantly alter laying performance during the trial, whereas it increased (p < 0.05) the total follicles compared to the Ctrl diet. The eggs from the hens fed the BA diet exhibited increased (p > 0.05) relative weight of eggshell and yolk color than those that consumed the Ctrl diet. There were no significant changes following BA treatment regarding the serum lipid profile. Dietary BA treatment reduced the total triglyceride in livers to different extents, resulting in the decreased diameter and area of vacuoles in liver tissues. The low-dose BA treatment decreased the mRNA levels of fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD), while promoting the expression of lipoprotein lipase (LPL) compared to the Ctrl group (both p < 0.05). Of note, the expressions of farnesoid X receptor (FXR), apical sodium-dependent bile acid transporter (ASBT), and ileum bile acid-binding protein (IBABP) were notably downregulated (p < 0.05) by the low-dose BA treatment. Dietary BA treatment had no apparent effects on laying performance, whereas it increased the follicle frequency, eggshell weight, and yolk color. Moreover, a diet containing 95.01 mg/kg of BAs depressed ileal BA resorption and hepatic fatty deposition by reducing lipogenesis and promoting lipolysis, which may have a beneficial effect on the liver in late-phase layers.

Zearalenone (ZEA), a mycotoxin produced mainly by fungi belonging to Fusarium species in foods and feeds, causes a serious hazard to humans and animals. Numerous studies have revealed that ingesting ZEA can disrupt the reproductive function and impair the reproductive process in animals. This experiment was to investigate the toxicological effect and the mechanism of ZEA exposure on reproduction in pigs during early stages of pregnancy. In the present study, we treated with 0 to 80 μmol/L ZEA for 12 or 24 h in trophoblast ectoderm (pTr) cells. The results showed that ZEA had significantly decreased cell proliferation (P

Replacing corn with different levels of wheat in the iso-energy and -protein diet of broilers and the impacts on growth performance and intestinal homeostasis of broilers under the condition of supplying the multienzyme complex were evaluated in this study. A total of 480 10-day-old male broilers were assigned randomly to the low-level wheat group (15% wheat and 35.18% corn), the medium-level wheat group (30% and 22.27%), and the high-level wheat group (55.77% wheat without corn) until 21 d. The different levels of wheat supplementation did not affect hepatic function, serum glycolipid profile, or bone turnover. The replacement of corn with 55% wheat in the diet of broilers increased the body weight at 21 d and feed intake during 10 to 21 d (both p < 0.05), with a comparable feed conversion ratio. Compared with the low-wheat group, the dietary addition of medium or high wheat levels notably increased the ratio of villus height to crypt depth in the duodenum (p < 0.05) and the ileal villus height (p < 0.05). Meanwhile, the supplementation of medium and high wheat in the diet increased the proportion of Bacteroidetes, and a diet with high wheat proportion elevated the content of Firmicutes when compared to the low-level wheat group (both p < 0.05). In addition, the diet containing 30–55% wheat enhanced the anti-inflammatory capability in both the ileum and the serum. These findings suggest that the replacement of corn with 55% wheat in the diet improved the growth performance of 21-day-old broilers, which might be linked to the alteration in intestinal morphology and cecal microbiota.

Abstract Inflammatory bowel disease (IBD), which includes Crohn's disease (CD) and ulcerative colitis (UC), has emerged as a global disease with high incidence, long duration, devastating clinical symptoms, and low curability (relapsing immune response and barrier function defects). Mounting studies have been performed to investigate its pathogenesis to provide an ever‐expanding arsenal of therapeutic options, while the precise etiology of IBD is not completely understood yet. Recent advances in high‐throughput sequencing methods and animal models have provided new insights into the association between intestinal microbiota and IBD. In general, dysbiosis characterized by an imbalanced microbiota has been widely recognized as a pathology of IBD. However, intestinal microbiota alterations represent the cause or result of IBD process remains unclear. Therefore, more evidences are needed to identify the precise role of intestinal microbiota in the pathogenesis of IBD. Herein, this review aims to outline the current knowledge of commonly used, chemically induced, and infectious mouse models, gut microbiota alteration and how it contributes to IBD, and dysregulated metabolite production links to IBD pathogenesis.

Amino acids serve not only as building blocks for proteins, but also as substrates for the synthesis of low-molecular-weight substances involved in hepatic lipid metabolism. In the present study, eighteen weaned female piglets at 35 days of age were fed a corn- and soybean meal-based diet containing 20%, 17%, or 14% crude protein (CP), respectively. We found that 17% or 20% CP administration reduced the triglyceride and cholesterol concentrations, while enhanced high-density lipoprotein cholesterol (HDL-C) concentration in serum. Western blot analysis showed that piglets in the 20% CP group had higher protein abundance of hormone-sensitive triglyceride lipase (HSL) and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), as compared with other groups. Moreover, the mRNA expression of sterol regulatory element binding transcription factor 1 (SREBPF1), fatty acid synthase (FASN), and stearoyl-CoA desaturase (SCD) were lower in the 17% or 20% CP group, compared with those of the piglets administered with 14% CP. Of note, the mRNA level of acetyl-CoA carboxylase alpha (ACACα) was lower in the 17% CP group, compared with other groups. Additionally, the mRNA level of lipoprotein lipase (LPL), peroxisome proliferator-activated receptor alpha α (PPARα), glucose-6-phosphatase catalytic subunit (G6PC), and phosphoenolpyruvate carboxykinase 1 (PKC1) in the liver of piglets in the 20% CP group were higher than those of the 14% CP group. Collectively, our results demonstrated that dietary CP could regulate hepatic lipid metabolism through altering hepatic lipid lipogenesis, lipolysis, oxidation, and gluconeogenesis.

L-proline (proline) is a key regulator of embryogenesis, placental development, and fetal growth. However, the underlying mechanisms that support the beneficial effects of proline are largely unknown. This study used porcine trophectoderm cell line 2 (pTr2) to investigate the underlying mechanisms of proline in cell proliferation and redox homeostasis. Cells were cultured in the presence of 0, 0.25, 0.50, or 1.0 mmol/L proline for an indicated time. The results showed that 0.5 and 1.0 mmol/L proline enhanced cell viability. These effects of proline (0.5 mmol/L) were accompanied by the enhanced protein abundance of p-mTORC1, p-p70S6K, p-S6, and p-4E-BP1. Additionally, proline dose-dependently enhanced the mRNA expression of proline transporters [solute carrier family (SLC) 6A20, SLC36A1, SLC36A2, SLC38A1, and SLC38A2], elevated proline concentration, and protein abundance of proline dehydrogenase (PRODH). Furthermore, proline addition (0.25 or 0.5 mmol/L) resulted in lower abundance of p-AMPKα when compared with a control. Of note, proline resulted in lower reactive oxygen species (ROS) level, upregulated mRNA expression of the catalytic subunit of glutamate–cysteine ligase (GCLC) and glutathione synthetase (GSS), as well as enhanced total (T)-GSH and GSH concentration when compared with a control. These data indicated that proline activates themTORC1 signaling and modulates the intracellular redox environment via enhancing proline transport.

Salmonella typhimurium infection is associated with gastrointestinal disorder and cellular injury in the liver of both humans and animals. Cinnamaldehyde, the main component of essential oil from cinnamon, has been reported to have anti-inflammatory, anti-oxidative, and anti-apoptotic effects. However, it remains unknown whether cinnamaldehyde can alleviate Salmonella typhimurium infection-induced liver injury in mice. In the present study, we found that cinnamaldehyde attenuated Salmonella typhimurium-induced body weight loss, the increase of organ (liver and spleen) indexes, hepatocyte apoptosis, and the mortality rate in mice. Further study showed that cinnamaldehyde significantly alleviated Salmonella typhimurium-induced liver injury as shown by activities of alanine transaminase, aspartate transaminase, and myeloperoxidase, as well as malondialdehyde. The increased mRNA level of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, and IFN-γ) and chemokines (CCL2 and CCL3) induced by Salmonella typhimurium were significantly abolished by cinnamaldehyde supplementation. These alterations were associated with a regulatory effect of cinnamaldehyde on TLR2, TLR4, and MyD88. 16S rDNA sequence analysis showed that Salmonella typhimurium infection led to upregulation of the abundances of genera Akkermansia, Bacteroides, Alistipes, Muribaculum, and Prevotellaceae UCG-001, and downregulation of the abundances of genera Lactobacillus, Enterorhabdus, and Eggerthellaceae (unclassified). These alterations were reversed by cinnamaldehyde supplementation. In conclusion, cinnamaldehyde attenuated the inflammatory response, oxidative stress, and apoptosis in the liver of Salmonella typhimurium-infected mice. Supplementation of cinnamaldehyde might be a preventive strategy to alleviate liver injury caused by Salmonella typhimurium infection in humans and animals.

Nitroethane (NE), 2-nitroethanol (NEOH), and 2-nitro-1-propanol (NPOH) were comparatively examined to determine their inhibitory actions on rumen fermentation and methanogenesis in vitro. Fermentation characteristics, CH4 and total gas production, and coenzyme contents were determined at 6, 12, 24, 48, and 72 h incubation time, and the populations of ruminal microbiota were analyzed by real-time PCR at 72 h incubation time. The addition of NE, NEOH, and NPOH slowed down in vitro rumen fermentation and reduced the proportion of molar CH4 by 96.7%, 96.7%, and 41.7%, respectively (p < 0.01). The content of coenzymes F420 and F430 and the relative expression of the mcrA gene declined with the supplementation of NE, NEOH, and NPOH in comparison with the control (p < 0.01). The addition of NE, NEOH, and NPOH decreased total volatile fatty acids (VFAs) and acetate (p < 0.05), but had no effect on propionate concentration (p > 0.05). Real-time PCR results showed that the relative abundance of total methanogens, Methanobacteriales, Methanococcales, and Fibrobacter succinogenes were reduced by NE, NEOH, and NPOH (p < 0.05). In addition, the nitro-degradation rates in culture fluids were ranked as NEOH (−0.088) > NE (−0.069) > NPOH (−0.054). In brief, the results firstly provided evidence that NE, NEOH, and NPOH were able to decrease methanogen abundance and dramatically decrease mcrA gene expression and coenzyme F420 and F430 contents with different magnitudes to reduce ruminal CH4 production.