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.

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.

Porcine bile acids promote the utilization of fat and vitamin A under low-fat diets.

Fat-soluble vitamin malabsorption may occur due to low dietary fat content, even in the presence of an adequate supply of fat-soluble vitamins. Bile acids (BAs) have been confirmed as emulsifiers to promote fat absorption in high-fat diets. However, there are no direct evidence of exogenous BAs promoting the utilization of fat-soluble vitamins associated with fat absorption in vitro and in vivo. Therefore, we chose laying hens as model animals, as their diet usually does not contain much fat, to expand the study of BAs. BAs were investigated in vitro for emulsification, simulated intestinal digestion, and release rate of fat-soluble vitamins. Subsequently, a total of 450 healthy 45-week-old Hy-Line Gray laying hens were chosen for an 84-day feeding trial. They were divided into five treatments, feeding diets supplemented with 0, 30, 60, 90, and 120 mg/kg BAs, respectively. No extra fat was added to the basic diet (crude fat was 3.23%). In vitro, BAs effectively emulsified the water-oil interface. Moreover, BAs promoted the hydrolysis of fat by lipase to release more fatty acids. Although BAs increased the release rates of vitamins A, D, and E from vegetable oils, BAs improved for the digestion of vitamin A more effectively. Dietary supplementation of 60 mg/kg BAs in laying hens markedly improved the laying performance. The total number of follicles in ovaries increased in 30 and 60 mg/kg BAs groups. Both the crude fat and total energy utilization rates of BAs groups were improved. Lipase and lipoprotein lipase activities were enhanced in the small intestine in 60, 90, and 120 mg/kg BAs groups. Furthermore, we observed an increase in vitamin A content in the liver and serum of laying hens in the 60, 90, and 120 mg/kg BAs groups. The serum IgA content in the 90 and 120 mg/kg BAs groups was significantly improved. A decrease in serum malondialdehyde levels and an increase in glutathione peroxidase activity were also observed in BAs groups. The present study concluded that BAs promoted the absorption of vitamin A by promoting the absorption of fat even under low-fat diets, thereupon improving the reproduction and health of model animals.

Dietary supplementation of porcine bile acids improves laying performance, serum lipid metabolism and cecal microbiota in late-phase laying hens.

Due to the exceptional laying performance of hens, the demand on lipid metabolism and oxidation in vivo is vigorous, resulting in excessive lipid accumulation in late-phase hens, which lowers the production performance. Bile acids regulate lipid metabolism and gut microbiota in humans and animals. However, the effect of porcine bile acids on lipid metabolism and cecal microbiota in laying hens in the late phase is still unclear. A total of 360 healthy 45-week-old laying hens were chosen for a 24-week feeding trial, where 0, 30, 60 and 90 mg/kg porcine bile acids were added to a basal diet, respectively. The results showed that dietary supplementation of 60 mg/kg bile acids increased egg production and feed conversion (P < 0.05). Also, 60 and 90 mg/kg porcine bile acids reduced abdominal fat percentage and body weight (P < 0.05). The levels of serum total cholesterol, triglyceride, and low-density lipoprotein cholesterol of hens decreased (P < 0.05) in bile acids supplement groups. As for cecal microbiota, bile acids supplementation did not affect the alpha diversity of cecal microbiota at the genus level. Moreover, dietary supplementation of 90 mg/kg bile acids resulted in an increase in the abundance of beneficial bacteria in the cecum, such as Lactobacillus, Bifidobacterium and Turicibacter. The changes in the cecal microbiota caused by bile acids supplementation correlated with serum lipid indexes. According to KEGG pathway analysis, dietary supplementation of 60 and 90 mg/kg bile acids promoted structural transformation of the cecal microbiota to down-regulate steroid biosynthesis, up-regulate fatty acid degradation and up-regulate unsaturated fatty acid biosynthesis. Meanwhile, bile acids bio-isomerization function of cecal microbiota was enhanced in 60 and 90 mg/kg bile acids treatment, and the short-chain fatty acid metabolism was also affected. In conclusion, the present study revealed dietary supplementation of porcine bile acids enriched probiotics in the gut and improved serum lipid metabolism of laying hens. These findings demonstrate that porcine bile acids can be a potential gut beneficial promoter for late-phase laying hens.

Effects of Zinc Methionine Hydroxy Analog Chelate on Laying Performance, Serum Hormone Levels, and Expression of Reproductive Axis Related Genes in Aged Broiler Breeders.

Inorganic zinc (Zn) supplements are commonly used in poultry feeds, but their low utilization results in the increase of Zn excretion. Thus, to provide a new perspective for the substitution of inorganic Zn, a novel Zn methionine hydroxy analog chelate (Zn-MHA) was studied in the present study to evaluate its effects on laying performance, serum hormone indexes and reproductive axis-related genes in broilers breeders. A total of 480 Hubbard breeders (56-week-old) were fed a basal diet (containing 27.81 mg Zn/kg) without Zn addition for 2 weeks, and then allocated to 4 groups with 6 replicates (each replicate consisting of 10 cages and 2 breeders per cage) for 10 weeks. Four treatment diets given to broiler breeders included the basal diet added with 25, 50, and 75 mg/kg of Zn-MHA and 100 mg/kg of Zn sulfate (ZnSO4). The laying rate, egg weight and feed conversation ratio increased in the 75 mg/kg Zn-MHA group compared to the ZnSO4 group. The eggshell thickness was not decreased with the addition of 50 mg/kg and 75 mg/kg Zn-MHA in the diet compared to the 100 mg/kg ZnSO4 group. There was a significant improvement in the reproductive performance of breeders in the 75 mg/kg Zn-MHA group, including the fertility and 1-day-old offspring weight. Besides, serum sex hormone levels including FSH and P4 increased significantly in 75 mg/kg Zn-MHA group. No significant effect on the ovarian weight or the number of follicles in broiler breeders was observed by supplementing Zn-MHA. Compared to the 100 mg/kg ZnSO4 group, dietary supplementation with 75 mg/kg of Zn-MHA showed an up-regulation of the FSHR mRNA in the granular layer of follicles. However, dietary supplementation of Zn-MHA had no effects on mRNA expressions of the ovarian LHR and PRLR genes. These findings reinforce the suggestion that Zn-MHA (75 mg/kg) could replace ZnSO4 (100 mg/kg) as a Zn supplement in diet of broiler breeders, which resulted in better laying and reproduction performances by regulating the expression levels of reproductive axis related genes and serum hormone levels.

Safety Evaluation of Porcine Bile Acids in Laying Hens: Effects on Laying Performance, Egg Quality, Blood Parameters, Organ Indexes, and Intestinal Development.

Bile acids (BAs) have long been known to facilitate digestion, transport, and absorption of lipids in the small intestine as well as regulate host lipid metabolic homeostasis. However, excessive BAs may lead to long-term damage to tissue. Also, it is unknown whether different levels of porcine BAs supplementation could improve performance, host metabolism, intestinal functions in laying hens. Hence, this study was aimed to investigate the potential effects of BAs addition on laying performance, egg quality, blood parameters, organ indexes, and intestinal histopathology of hens in the late phase. A total of 300 58-week-old Hy-line Gray hens were randomly divided into five groups which fed a basal diet (control) or basal diets supplemented with 60, 300, 600, and 3,000 mg/kg BAs for 56 days. Compared with the control group, no significant differences (P > 0.05) were observed in egg production, egg weight, ADFI, and FCR of hens in 60, 300, 600, and 3,000 mg/kg BAs groups. Dietary 60 mg/kg BAs supplementation resulted in a significant increase (P < 0.05) in egg mass. Meanwhile, no significant differences were observed in egg quality, including eggshell strength, eggshell thickness, albumen height, and Haugh unit among any treatment groups (P > 0.05). Dramatically, dietary 3,000 mg/kg BAs supplement decreased yolk color (P < 0.05). There was no significant difference in the blood parameters such as WBC, RBC, HGB, HCT, and PLT among any treatments. However, in 3,000 mg/kg BAs group, ovary coefficient was lower than the control (P < 0.05), and serum urea and creatinine were higher than the control (P < 0.05). Also, kidney and oviduct injury appeared in 3,000 mg/kg BAs group. These results indicated that a porcine BAs concentration of 3,000 mg/kg may cause harmful effects while 600 mg/kg was non-deleterious to laying hens after a daily administration for 56 days, namely that dietary supplement of up to 10 times the recommended dose of BAs was safely tolerated by 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.

Effects of the Agaricus bisporus stem residue on performance, nutrients digestibility and antioxidant activity of laying hens and its effects on egg storage.

OBJECTIVE: The purpose of this experiment was to investigate the effects of the Agaricus bisporus stem residue (ABSR) on the performance, nutrients digestibility, antioxidant activity of laying hens, and its effects on egg storage to determine the appropriate dosage of ABSR, so as to provide a scientific basis for the effective utilization of ABSR. METHODS: A total of 384 53-wk-old Nongda III layers were randomly divided into six treatments, four replicates in each treatment and 16 birds in each replicate. The control treatment was fed with basic diet, while experimental treatments were fed with diets of 2%, 4%, 6%, 8%, and 10% ABSR respectively. The experimental period was 56 d. RESULTS: The results showed that compared with the control treatment, ABSR had no significant effect on laying performance (p>0.05). The crude protein and total energy digestibility of experimental treatments was significantly higher than those of control treatment (p<0.05). When eggs were stored for 1 wk, 2 wk, and 3 wk at 25°C, there were no significant differences in egg storage between the experimental treatments and the control treatment (p>0.05). The superoxide dismutase (SOD) activity and glutathione peroxidase (GSH-Px) activity in the serum of the experimental treatments were significantly higher than those of the control treatment (p<0.05), and the malonaldehyde (MDA) content did not change dramatically. SOD activity in yolk of experimental treatments was significantly higher than that in control treatment (p<0.05); MDA content in yolk was markedly lower than that in control treatment (p<0.05). The activity of GSH-Px and SOD in yolk of experimental treatments was significantly higher than that of control treatment stored at 25°C for 21 d, and the content of MDA was significantly lower than that of control treatment (p<0.05). CONCLUSION: ABSR can be used to improve the antioxidant activity of laying hens without affecting laying performance.

The dynamic changes of interferon lambdas related genes and proteins in JAK/STAT pathway in both acute and chronic HIV-1 infected patients.

BACKGROUND: Host immune responses during acute HIV-1 infection can influence the viral setpoint, which is a predictor of disease progression. Interferon (IFN)-lambdas are newly classified type III interferons, which use JAK-STAT pathway. Currently, the dynamics of IFN-lambdas related genes and proteins expression in the signaling pathway have not been well elaborated, especially in acute HIV-1-infected patients. OBJECTIVES: To evaluate the dynamic changes of IFN-lambdas related genes and proteins in JAK/STAT pathway in acute HIV-1-infected patients, and analyze their correlation with CD4 T cell counts and HIV-1 viral loads. STUDY DESIGN: Real-time PCR and flow cytometry methods were used to evaluate the dynamic changes of IFN-lambdas related genes and proteins in JAK/STAT pathway in both acute and chronic HIV-1-infected patients. RESULTS: The IFN-alpha receptors (R), IFN-gamma R, IFN-lambdas R and STAT1 mRNA and protein levels increased in acute HIV-1-infected patients (p < 0.01), in addition, Mx1 mRNA levels in acute HIV-1-infected patients are higher than those in HIV-negative subjects. IFN-lambdas R and IFN-alpha R mRNA levels are inversely correlated with CD4+ T-cell counts, but are positively correlated with viral loads. CONCLUSIONS: The dynamic changes of IFNs related genes in JAK-STAT pathway in acute HIV-1 infection will deepen our understanding of the roles of IFN-lambdas in HIV pathogenesis.

Perturbations of Monocyte Subsets and Their Association with T Helper Cell Differentiation in Acute and Chronic HIV-1-Infected Patients.

Monocytes have been recently subdivided into three subsets: classical (CD14++CD16-), intermediate (CD14++CD16+), and non-classical (CD14+CD16++) subsets, but phenotypic and functional abnormalities of the three monocyte subsets in HIV-1 infection have not been fully characterized, especially in acute HIV-1 infection (AHI). In the study, we explored the dynamic changes of monocyte subsets and their surface markers, and the association between monocyte subsets and the IFN-γ, interleukin (IL)-4, IL-17, and TNF-α producing CD4+ T cells in acute and chronic HIV-1-infected patients. We found that, in the acute HIV-1-infected individuals, the frequency of the intermediate CD14++CD16+ monocyte subsets, the CD163 density and HLA-DR density on intermediate CD14++CD16+ monocytes, and plasma soluble form of CD163 (sCD163) were significantly higher than that in healthy controls. Intermediate CD14++CD16+ monocyte subsets and their HLA-DR expression levels were inversely correlated with the CD4+ T cell counts, and the intermediate CD14++CD16+ monocytes were positively correlated with plasma sCD163. In contrast to the non-classical CD14+CD16++ and classical CD14++CD16- monocyte subsets, the frequency of the intermediate CD14++CD16+ monocytes was positively associated with the frequency of IFN-γ and IL-4 producing CD4+ T cells in HIV-1-infected patients. Taken together, our observations provide new insight into the roles of the monocyte subsets in HIV pathogenesis, particularly during AHI, and our findings may be helpful for the treatment of HIV-related immune activation.

Cytokine levels contribute to the pathogenesis of minimal hepatic encephalopathy in patients with hepatocellular carcinoma via STAT3 activation.

Hepatocellular carcinoma (HCC) patients were grouped according to the degree of encephalopathy, with healthy volunteers as controls. We investigated clinical presentation, protein and mRNA expression of 14 cytokines, and activation of six STAT proteins, the downstream signaling mediators. Levels of all 14 cytokines were significantly elevated in HCC patients with clinical hepatic encephalopathy. Statistical analysis showed that levels of IL-1ß, IL-6, IFNγ, IL-17α, IFNλ2 and IFNλ3 were correlated with minimal hepatic encephalopathy (MHE). Multivariate regression analysis identified serum IL-6, IFNλ3 and IL-17α as independent risk factors for MHE. Increased mRNA levels of IL-6 and IFNγ were associated with MHE. Among the STAT proteins examined, only STAT3 was elevated in MHE. Treatment with a STAT3 inhibitor protected neurons from cytokine-induced apoptosis in vitro. In conclusion, this study identified potential biomarkers for MHE in HCC. The cytokines investigated may induce neural apoptosis via STAT3 in the pathogenesis of MHE in HCC.

Clinical correlation between HBV infection and concomitant bacterial infections.

Bacterial infections are common in patients suffering viral hepatitis and critical for prognosis. However, any correlation between HBV and concomitant bacterial infections is not well characterized. A retrospective study was conducted from Jan 2012 to Jan 2014 on 1333 hospitalized patients infected with bacteria. Among them, 491 HBV-infected patients were co-infected with E. coli (268), S. aureus (61), P. aeruginosa (64) or K. pneumoniae (98). A group of 300 complication-free chronically HBV-infected patients were controls. We found that HBV DNA levels were elevated in patients with each of the bacterial infections (all P < 0.05). ALT and HBeAg were strong determinants of high HBV DNA concentration. Patterns of determinants varied in infections by Gram-positive and Gram-negative bacteria. Patients with HBV DNA ≥ 2000 IU/mL had higher rates of all four concomitant bacterial infections (all P < 0.001). All types of strains isolated from HBV-positive patients showed less resistance to tested antimicrobials. The HBV DNA serum concentrations were inversely correlated to the number of ineffective antimicrobials in E. coli, P. aeruginosa and K. pneumoniae infections (P = 0.022, 0.017 and 0.016, respectively), but not S. aureus (P = 0.194). In conclusion, bacterial infections are associated with a high level of HBV replication, which, in turn, has a significant positive impact on bacterial resistance to antimicrobials. These correlations vary between Gram-negative and Gram-positive bacteria.

Mechanism of blood coagulation in common carp (Cyprinus carpio).

In vitro, carp blood was anticoagulated by using MgSO(4) at a final concentration of 22.2 mmol L(-1) and sodium citrate at a final concentration of 11.8 mmol L(-1) . The coagulation times for carp plasma diluted by ion-free water (1:1), and that of carp plasma to which thrombocytes and small lymphocytes were added, were measured at 23 °C using standard methods, and then contrasted with the coagulation times for plasma obtained from chickens and rabbits. The shapes of the thrombocytes and small lymphocytes, which were either wet mounted or stained with hematoxylin and eosin, were observed under a light microscope. We found that: (i) the coagulation reaction of carp blood was significantly (P < 0.01) accelerated by the addition of ion-free water; (ii) the three types of blood cells (thrombocytes, small lymphocytes and red blood cells) promoted plasma coagulation to a similar extent (P > 0.05); (iii) in carp Mg(2+) plasma and K(2) C(2) O(4) plasma, the thrombocytes were usually morphologically normal, but many small lymphocytes were destroyed and became aggregated; (iv) in the citrate plasma, thrombocytes were often aggregated, but the small lymphocytes were usually morphologically normal; and (v) the coagulation time for chicken and rabbit plasma was significantly extended by adding ion-free water.