The impact of NUMB on chicken abdominal adipogenesis: A comprehensive analysis.

Excessive abdominal fat deposition negatively impacts poultry meat production and carcass yield. Identification of novel adipogenesis regulators may help improve production performance declines caused by excessive fat deposition. NUMB Endocytic Adaptor Protein (NUMB) typically functions as a cell fate determinant and plays a significant role in cell development and various diseases. Here, we found that NUMB is abundantly expressed in chicken abdominal fat depots and is induced in cultured adipocytes following adipogenic treatment. The gain- and loss-of-function experiments demonstrated that NUMB promotes the proliferation and G1/S transition of chicken adipocytes, enhances adipocyte differentiation, and increases the expression of PPARγ1 transcript. Through mRNA-seq analysis and molecular experiments, we further confirmed that NUMB inhibits the transcriptional activation of the NOTCH1 pathway and the expression of the downstream transcription factor HES1 by inducing NOTCH1 degradation. Nevertheless, the inhibition of the NOTCH1/HES1 axis alone cannot fully explain NUMB's role in adipogenesis, as NUMB also regulates the expression of multiple adipogenic transcription factors such as E2F1, EGR2, and NR4A3. Our data suggest that NUMB is a potent activator of adipogenesis and enhances our understanding of its regulatory mechanisms in chicken abdominal fat deposition.

Integrated transcriptomic and metabolomic analyses reveal potential regulatory pathways regulating bone metabolism pre- and postsexual maturity in hens.

At the onset of sexual maturity, the increasing circulating estrogen stimulates the formation of medullary bone, which provides available calcium for eggshell formation. The bone loss of laying hens is caused by the continuous dynamic changes of structure bone leading to bone fragility and susceptibility. The degree of medullary bone mineralization in sexual maturity is positively correlated with bone quality in the late laying stage. This study aimed to explore the molecular regulation mechanism of bone metabolism pre- and postsexual maturity in hens based on the joint analysis of transcriptome and metabolome. A total of 50 Hy-line Sonia pullets with comparable body weight at 13 wk were selected. Eight pullets were killed at 15 wk (juvenile hens, JH) and 19 wk (laying hens, LH), and LHs were killed within 3 h after oviposition. Differentially expressed genes and metabolites in tibia were analyzed based on transcriptome and metabolome, and then combined to construct the relevant metabolisms and hub genes. In the LH hens, plasma levels of estrogen and tartrate-resistant acid phosphatase were significantly elevated by 1.7 and 1.3 times. In addition, the midpoint diameter, bone mineral density and bone mineral content of the tibia and femur were higher at 19 wk of age. A total of 580 differentially expressed genes were found between the JH and LH group in the tibia, including 280 up-regulated, and 300 down-regulated genes in the LH group. Gene set enrichment analysis (GSEA) showed that the intracellular biosynthesis and secretion of matrix vesicles were significantly enrichment in the LH hens. A total of 21 differential metabolites were identified between JH and LH group. Estradiol valerate positively correlated with L-theanine, tryptophan betaine, dopamine, and perindopril. Joint analysis showed that the top 20 hub genes were enriched in cholesterol biosynthesis and phospholipid metabolism, which played a key regulatory role in bone metabolism during pre- and postsexual maturity. These results provide a theoretical foundation for maintaining efficient egg production and reducing bone health problems in laying hens.

Identification of ID1 and miR-150 interaction and effects on proliferation and apoptosis in ovine granulosa cells.

Granulosa cells (GCs) proliferation and apoptosis play a significantly role in follicular development and atresia. ID1 and miR-150 are involved in cell apoptosis and follicular atresia, but the interaction and function of ID1 and miR-150 in GCs are unclear. This study focuses on ID1 and miR-150 in terms of the interaction and effects on proliferation and apoptosis in ovine granulosa cells. Our findings revealed that ID1 decreased the promoter activity and expression level of oar-miR-150. However, the expression of ID1 was downregulated by miR-150, and ID1 was identified as a target gene of oar-miR-150. miR-150 mimic inhibited proliferation and upregulated apoptosis rate in ovine GCs, while the results of miR-150 inhibitor were opposite. Overexpression of ID1 significantly inhibited ovine GCs proliferation and cell cycle-related genes (CDK1, CDK2, CDK4, CCND2, CDC20, and PCNA) expression, whereas knockdown of ID1 promoted cell proliferation and those genes expression. Overexpression of ID1 significantly downregulated mitochondrial membrane potential and Bcl-2 expression in ovine GCs, and upregulated the expression of pro-apoptosis genes Bax, Caspase-3, and Caspase-9, whereas the results of ID1 knockdown were reversed. Collectively, these findings indicate the interaction and the vital role of ID1 and miR-150 on proliferation and apoptosis in ovine granulosa cells, which may suggest a novel target for ovine follicular development and atresia.

[Effect of the chicken zp1 gene on osteoblast mineralization].

The aim of this study was to clone the chicken zp1 gene encoding zona pellucida 1 (Zp1) and investigate its tissues expression profile and its effect on osteoblast mineralization. The expression level of zp1 was quantified in various tissues of laying hens and in the tibia of the pre- and post-sexual maturity by RT-qPCR. Zp1 overexpressed vector was transfected into chicken calvarial osteoblasts which were induced differentiation for 8 days, and the extracellular mineral and the expression of mineralization-related genes were detected. The full-length chicken zp1 gene is 3 045 bp, encoding 958 amino acids residuals, and has two N-glycosylation sites. The highest expression level of the zp1 gene was found in the liver, followed by the tibia and yolk membrane, while no expression was detected in the heart and eggshell gland. Compared with the pre-sexual maturity hens, the concentration of estrogen (E2) in plasma, the content of glycosaminoglycan (GAG) and the expression level of the zp1 gene in the tibia with post-sexual maturity were higher. The extracellular matrix and the level of osteoblast mineralization-related genes showed a significantly upregulated expression in chicken calvarial osteoblasts with Zp1 overexpressed and addition of estrogen. The expression of the zp1 gene is tissue-specific and positively regulated osteoblast mineralization under the action of estrogen, laying the foundation for elucidating the functional properties of Zp1 in chicken bones during the egg production period.

Malfunctioned inflammatory response and serotonin metabolism at the microbiota-gut-brain axis drive feather pecking behavior in laying hens.

Feather pecking (FP) is a multifactorial abnormal behavior in laying hens where they display harmful pecks in conspecifics. FP has been associated with the altered functioning of the microbiome-gut-brain axis affecting host emotions and social behavior. The altered levels of serotonin (5-HT), a key monoaminergic neurotransmitter at both terminals of the gut-brain axis, affect the development of abnormal behavior, such as FP in laying hens. However, the underlying mechanism involving reciprocal interactions along the microbiota-gut-brain axis, particularly about the metabolism of 5-HT, remains unclear in FP phenotypes. This study examined the microbiota diversity, intestinal microbial metabolites, inflammatory responses, and 5-HT metabolism in divergently selected high (HFP; n = 8) and low (LFP; n = 8) FP hens to investigate the possible interconnections between FP behavior and the examined parameters. The 16S rRNA analysis revealed that compared to LFP birds, the gut microbiota of HFP birds exhibited a decrease in the abundance of phylum Firmicutes and genera Lactobacillus, while an increase in the abundance of phylum Proteobacteria and genera Escherichia Shigella and Desulfovibrio. Furthermore, the intestinal differential metabolites associated with FP phenotypes were mainly enriched in the tryptophan metabolic pathway. HFP birds had higher tryptophan metabolites and possibly a more responsive immune system compared to the LFP birds. This was indirectly supported by altered TNF-α levels in the serum and expression of inflammatory factor in the gut and brain. Moreover, HFP birds had lower serum levels of tryptophan and 5-HT compared to LFP birds, which was consistent with the downregulation of 5-HT metabolism-related genes in the brain of HFP birds. The correlation analysis revealed that genera Lactobacillus and Desulfovibrio were associated with differences in intestinal metabolites, 5-HT metabolism, and inflammatory response between the LFP and HFP birds. In conclusion, differences in the cecal microbiota profile, immune response and 5-HT metabolism drive FP phenotypes, which could be associated with the gut abundance of genera Lactobacillus and Desulfovibrio.

Melatonin alleviates ovarian function damage and oxidative stress induced by dexamethasone in the laying hens through FOXO1 signaling pathway.

Oxidative stress can trigger follicular atresia, and decrease follicles quantity in each development stage, thereby alleviating reproductive activity. The induction of oxidative stress in chickens through intraperitoneal injection of dexamethasone is a reliable and stable method. Melatonin has been shown to mitigate oxidative stress in this model, but the underlying mechanism remains unclear. Therefore, this study aimed to investigate whether melatonin can recover aberrant antioxidant status induced by dexamethasone and the specific mechanism behind melatonin-dependent protection. A total of 150 healthy 40-wk-old Dawu Jinfeng laying hens with similar body weights and laying rates were randomly divided into three groups, with five replicates per group and 10 hens per replicate. The hens in the control group (NS) received intraperitoneal injections of normal saline for 30 d, the dexamethasone group (Dex+NS) received 20 mg/kg dose of dexamethasone for the first 15 d, followed by the 15 d of normal saline treatment. While in the melatonin group (Dex+Mel), dexamethasone (20 mg/kg dose) was injected intraperitoneally in the first 15 d, and melatonin (20 mg/kg/d) was injected in the last 15 d. The results showed that dexamethasone treatment significantly enhanced oxidative stress (P < 0.05), while melatonin not only inhibited the oxidative stress but also notably enhanced the antioxidant enzymes superoxide dismutase (SOD), catalase activity (CAT), glutathione peroxidase (GSH-Px), and antioxidant genes CAT, superoxide dismutase 1 (SOD1), glutathione peroxidase 3 (GPX3), and recombinant peroxiredoxin 3 (PRDX3) expression (P < 0.05). Melatonin treatment also markedly reduced 8-hydroxy deoxyguanosine (8-OHdG), malondialdehyde (MDA), and reactive oxygen species (ROS) levels (P < 0.05) and apoptotic genes Caspase-3, Bim, and Bax in the follicle. In the Dex+Mel group, the Bcl-2 and SOD1 protein levels were also increased (P < 0.05). Melatonin inhibited the forkhead Box Protein O1 (FOXO1) gene and its protein expression (P < 0.05). In general, this investigation revealed that melatonin might decrease oxidative stress and ROS by enhancing antioxidant enzymes and genes, activating the antiapoptotic genes, and inhibiting the FOXO1 pathway in laying hens.

Restorative effects of Lactobacillus rhamnosus LR-32 on the gut microbiota, barrier integrity, and 5-HT metabolism in reducing feather-pecking behavior in laying hens with antibiotic-induced dysbiosis.

The development of abnormal feather-pecking (FP) behavior, where laying hens display harmful pecks in conspecifics, is multifactorial and has been linked to the microbiota-gut-brain axis. Antibiotics affect the gut microbial composition, leading to gut-brain axis imbalance and behavior and physiology changes in many species. However, it is not clear whether intestinal dysbacteriosis can induce the development of damaging behavior, such as FP. The restorative effects of Lactobacillus rhamnosus LR-32 against intestinal dysbacteriosis-induced alternations need to be determined either. The current investigation aimed to induce intestinal dysbacteriosis in laying hens by supplementing their diet with the antibiotic lincomycin hydrochloride. The study revealed that antibiotic exposure resulted in decreased egg production performance and an increased tendency toward severe feather-pecking (SFP) behavior in laying hens. Moreover, intestinal and blood-brain barrier functions were impaired, and 5-HT metabolism was inhibited. However, treatment with Lactobacillus rhamnosus LR-32 following antibiotic exposure significantly alleviated the decline in egg production performance and reduced SFP behavior. Lactobacillus rhamnosus LR-32 supplementation restored the profile of the gut microbial community, and showed a strong positive effect by increasing the expression of tight junction proteins in the ileum and hypothalamus and promoting the expression of genes related to central 5-HT metabolism. The correlation analysis revealed that probiotic-enhanced bacteria were positively correlated, and probiotic-reduced bacteria were negatively correlated with tight junction-related gene expression, and 5-HT metabolism, and butyric acid levels. Overall, our findings indicate that dietary supplementation with Lactobacillus rhamnosus LR-32 can reduce antibiotic-induced FP in laying hens and is a promising treatment to improve the welfare of domestic birds.

Transcriptomic analysis reveals the molecular mechanisms underlying osteoclast differentiation in the estrogen-deficient pullets.

Several previous reports have suggested that estrogen (E2) is a vital signal responsible for the regulation of skeletal homeostasis and bone remodeling in mammals. E2 could efficiently accelerate the growth of medullary bone in pullets during sexual maturity. Furthermore, the low E2 level can strengthen the mechanical bone functions in female hens. However, mechanistic studies to describe the effects of E2 on bone in pullets during the initiation of the puberty period are remaining elusive. Therefore, the aim of this study was to explore the effect of inhibiting E2 biosynthesis on the biomechanical properties and its molecular mechanism during sexual maturity of pullets. In this study, a total of 90 Hy-line Sonia pullets with comparable body weight at 13 wk of age were selected and categorized into 2 separate groups. Daily, 0.5 mg/4 mL of letrozole (LZ) was orally administered to the treatment (TRT) group and 4 mL of saline to the control (CON) group of pullets for 6 wk. Compared with the CON group, a lower plasma E2 level was observed in the TRT group. Furthermore, plasma P, Gla protein (BGP), and 1,25-dihydroxy vitamin D3 (1,25-(OH)2D3) levels were markedly suppressed, whereas the plasma alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) levels were significantly elevated. Moreover, the cortical bone thickness and breaking strength of the tibia and femur, the bone mineral density of the humerus, and the bone mineral content of the humerus as well as the femur were increased significantly. The expression levels of 340 differentially expressed genes (DEGs) differed significantly between the CON and TRT group in the tibia at 19 wk of age. Among them, 32 genes were up-regulated, whereas 308 were down-regulated in the TRT group. The variations in candidate genes associated with osteoclast differentiation and cell adhesion may indicate that LZ inhibits E2 biosynthesis, consequently, reduces osteoclast differentiation by suppressing inter-cellular communication and cells attaching to extracellular matrix components. Taken together, the present study demonstrated that inhibiting E2 synthesis during sexual maturity of pullets decreased osteoclast differentiation and considerably enhanced bone quality.

Transcriptome profile reveals novel candidate genes associated with bone strength in end-of-lay hens.

Bone weakness causes many problems such as osteoporosis, bone fractures, and economic loss, especially at the late stage of lay, in laying hen production. However, the genetic factors and molecular mechanism affecting the bone strength is still largely unknown. To elucidate the molecular mechanism and genetic factors affecting bone strength, a total of six cDNA libraries were constructed and used to compare genetic differences between tibia with higher（Group HBS）and lower（Group LBS）breaking strength in Hyline grey layers. A comparison between Groups HBS and LBS revealed nine differentially expressed genes, of which five were upregulated and four were downregulated in the LBS relative to the HBS in tibia. Our results showed novel candidate genes concerned with bone strength in the late laying period. These include transcription factor paired box protein Pax-5 (Pax5), tissue inhibitor of Metallopoteinase-4 (TIMP4), Kelch-like protein 14 (KLHL14), predicted MAGUK p55 subfamily member 7 isoform X4 (MPP7) and Osteoclast-associated Ig-like receptor (OSCAR). Our data provide a vital resource for discovering important candidate genes associated with bone strength and will help further study the molecular mechanisms for bone remodeling.

Whole blood gas and biochemical reference intervals for Lohmann Silver layers.

The blood gas and biochemical reference range established with i-STAT clinical analyzer in avian has become common, however, the reference value for various laying hen lines is limited. Therefore, blood gas and biochemical reference intervals will be established for Lohmann Silver layers in the pre- and post-laying periods. The blood sample was collected at a 4-wk interval. A total of 230 Lohmann Silver layers including 80 pullets (5-17 wk) and 150 laying hens (21-37 wk) were collected for whole blood measurement with the i-STAT clinical analyzer. The CG8+ cartridge provides values of the following 13 parameters: sodium (Na mmol/L), potassium (K mmol/L), ionized calcium (iCa mmol/L), glucose (Glu mg/dL), hematocrit (Hct% Packed Cell Volume [PCV]), pH, partial pressure carbon dioxide (PCO2 mm Hg), partial pressure oxygen (PO2 mm Hg), total concentration carbon dioxide (TCO2 mmol/L), bicarbonate (HCO3 mmol/L), base excess (BE mmol/L), oxygen saturation (sO2%), and hemoglobin (Hb g/dL). The correlation of these parameters and the effect of physiological status were investigated. The reference value interval was established with a reference value advisor for pre-laying and post-laying birds. Correlations were found to be statistically significant, especially between BE and HCO3 and TCO2. Besides, values in Na, iCa, K, Hct, Hb, sO2 differed significantly between the pre- and post-laying periods. Data in this study might serve as important information for facilitating the genetic selection and assessing the health of Lohmann Silver laying hens.

Effect of in ovo injection of serotonin on the behavior and hormone level in laying hens.

Feather pecking is a typically abnormal behavior that significantly impacts breeding efficiency and animal welfare in the egg production sector. Serotonin (5-HT) is essential for neuronal development and behavioral regulation. This study evaluated the effects of birds' behavioral development (including feather pecking) and changes in serum hormones in chickens followed in ovo injection of 5-HT. On day 11, incubated eggs were injected with 5-HT at 0 (saline control), 5 ug (low) or 15 ug (high) (n = 166 per treatment). The hatched female chicks were raised under similar conditions up to 20 weeks of age (n = 60 per treatment). Birds' behaviors were recorded using a digital video recording system. The time to first vocalize and first move, along with the duration of vocalization and escape attempts during the isolation test, during isolation test were analyzed on day 1, and week 4, 8, 12, 16 and 20. Blood samples were collected followed behavioral tests (n = 5/treatment). The expression of 5-HTR1A genes in the hypothalamus was measured by real-time PCR. Compared to controls, 5-HT administrated pullets had greater body weight (P < 0.05) with an improved feed conversion rate (P < 0.05) as well as higher serum concentrations of norepinephrine (NE) regardless of their doses. In addition, serum dopamine (DA) concentrations were lower in both high- and low-dose pullets at 8 and 12 weeks of age (P < 0.05). Also, a decrease in fearfulness response was observed based on the test to vocalize and duration of vocalization (P < 0.05). Further, this exhibited a lesser frequency of total aggressive behavior compared with the chicks in the control group, especially at 8 weeks of age (P < 0.05), where it is associated with elevated serum 5-HT concentration and upregulated hypothalamic expression of 5-HTR1A (P < 0.05). The changes of these hormone concentrations and gene expressions suggested that 5-HT accumulation in early embryonic stages may alter both the adrenergic and serotonergic systems, which could further regulate the isolation behavior and improve birds' growth performance to a certain extent.

Integrated analysis of miRNA-mRNA interaction in ovaries of Turpan Black Sheep during follicular and luteal phases.

To investigate the regulatory mechanism of the follicular-luteal phase transition in Turpan black sheep (Ovis aries), the genome-wide expression patterns of microRNAs (miRNAs) and genes were investigated in ovaries of six sheep (3 years and single lamb with 3 consecutive births) during follicular and luteal phases of the oestrous cycle. Bioinformatic analysis was used to screen potential miRNAs and genes related to Turpan black sheep ovarian function. RT-qPCR was used to validate the sequencing results. In total, we identified 139 known and 71 novel miRNAs in the two phases with miRNA-seq, and a total of 19 miRNAs were significantly differentially expressed, of which 7 were up-regulated and 12 were down-regulated in the follicular phase compared with luteal phase. A total of 150 genes were significantly differentially expressed, including 63 up-regulated and 87 down-regulated in the follicular phase compared with the luteal phase by RNA-seq data analysis. Those DEGs were significantly enriched in 103 GO terms and several KEGG pathways, including metabolic pathway, ovarian steroidogenesis, steroid hormone biosynthesis and oestrogen signalling pathway. In addition, we created a miRNA-mRNA regulatory network to further elucidate the mechanism of follicular-luteal transition. Finally, we identified key miRNAs and genes including miR-143, miR-99a, miR-150, miR-27a, miR-125b, STAR, STAT1, which might play crucial roles in reproductive hormone biosynthesis and follicular development. The miRNA-mRNA interactive network clearly illustrates molecular basis involving in follicular-luteal transition.

Melatonin regulates chicken granulosa cell proliferation and apoptosis by activating the mTOR signaling pathway via its receptors.

Melatonin is a key regulator of follicle granular cell maturation and ovulation. The mammalian target of rapamycin (mTOR) pathway plays an important role in cell growth regulation. Therefore, our aim was to investigate whether the mTOR signaling pathway is involved in the regulation of melatonin-mediated proliferation and apoptotic mechanisms in granulosa cells. Chicken follicle granular cells were cultured with melatonin (0, 2, 20, or 200 µmol/L) for 48 h. The results showed that melatonin treatment enhanced proliferation and suppressed apoptosis in granular cells at 20 µmol/L and 200 µmol/L (P < 0.05) by upregulation of cyclin D1 (P < 0.01) and Bcl-2 (P < 0.01) and downregulation of P21, caspase-3, Beclin1, and LC3-II (P < 0.01). The effects resulted in the activation of the mTOR signaling pathway by increasing the expression of avTOR, PKC, 4E-BP1, S6K (P < 0.05), p-mTOR, and p-S6K. We added an mTOR activator and inhibitor to the cells and identified the optimal dose (10 µmol/L MHY1485 and 100 nmol/L rapamycin) for subsequent experiments. The combination of 20 µmol/L melatonin and 10 µmol/L MHY1485 significantly enhanced granulosa cell proliferation (P < 0.05), while 100 nmol/L rapamycin significantly inhibited proliferation and enhanced apoptosis (P < 0.05), but this action was reversed in the 20-µmol/L melatonin and 100-nmol/L rapamycin cotreatment groups (P < 0.05). This was confirmed by mRNA and protein expression that was associated with proliferation, apoptosis, and autophagy (P < 0.05). The combination of 20 µmol/L melatonin and 10 µmol/L MHY1485 also activated the mTOR pathway upstream genes PI3K, AKT1, and AKT2 and downstream genes PKC, 4E-BP1, and S6K (P < 0.05), as well as protein expression of p-mTOR and p-S6K. Rapamycin significantly inhibited the mTOR pathway-related genes mRNA levels (P < 0.05). In addition, activation of the mTOR pathway increased melatonin receptor mRNA levels (P < 0.05). In conclusion, these findings demonstrate that melatonin regulates chicken granulosa cell proliferation and apoptosis by activating the mTOR signaling pathway via its receptor.