Integration of Whole-Genome Resequencing and Transcriptome Sequencing Reveals Candidate Genes in High Glossiness of Eggshell.

Eggshell gloss is an important characteristic for the manifestation of eggshell appearance. However, no study has yet identified potential candidate genes for eggshell gloss between high-gloss (HG) and low-gloss (LG) chickens. The aim of this study was to perform a preliminary investigation into the formation mechanism of eggshell gloss and to identify potential genes. The eggshell gloss of 300-day-old Rhode Island Red hens was measured from three aspects. Uterine tissues of the selected HG and LG (n = 5) hens were collected for RNA-seq. Blood samples were also collected for whole-genome resequencing (WGRS). RNA-seq analysis showed that 150 differentially expressed genes (DEGs) were identified in the uterine tissues of HG and LG hens. These DEGs were mainly enriched in the calcium signaling pathway and the neuroactive ligand-receptor interaction pathway. Importantly, these two pathways were also significantly enriched in the WGRS analysis results. Further joint analysis of WGRS and RNA-seq data revealed that 5-hydroxytryptamine receptor 1F (HTR1F), zinc finger protein 536 (ZNF536), NEDD8 ubiquitin-like modifier (NEDD8), nerve growth factor (NGF) and calmodulin 1 (CALM1) are potential candidate genes for eggshell gloss. In summary, our research provides a reference for the study of eggshell gloss and lays a foundation for improving egg glossiness in layer breeding.

Ligilactobacillus Salivarius improve body growth and anti-oxidation capacity of broiler chickens via regulation of the microbiota-gut-brain axis.

Certain strains of probiotic bacteria can secret functional substances namely digestive enzymes and functional peptides to regulate physiological conditions such as digestion and anti-oxidation, which are often incorporated in industrial broiler chick production. However, few studies have detailed the action mechanisms and effects of these bacteria on regulating growth and anti-oxidation levels in broiler chickens. Ligilactobacillus salivarius is a strain of probiotic bacteria used as dietary supplement. In the present study, Ligilactobacillus salivarius was evaluated for its secreted digestive enzymes in vitro. To detailed evaluate the action mechanisms and effects of gastrointestinal tract (GIT) microbiota on alleviating anti-oxidation levels of broiler chickens through the gut-brain axis. Ligilactobacillus salivarius was cultured and supplemented in the food of broilers to evaluate the probiotic effect on growth and anti-oxidation by modulation of gut microbial composition and its functional metabolites using metagenomic and metabolomic assays. Biochemical results showed that Ligilactobacillus salivarius secreted digestive enzymes: protease, lipase, and amylase. Broiler chickens with Ligilactobacillus salivarius supplemented for 42 days, showed increased body weights, a reduced oxidative status, decreased malondialdehyde levels, and improved activities rates of total superoxide dismutase, glutathione peroxidase IIand IV improved. The microbial composition of caecum was more abundant than those broiler without probiotics supplementation, owing 400 of total number (489) of bacterial operational taxonomic units (OTU). The genera of Lactobacillus, Megamonas, Ruminoccoccaceae, Ruminococcus, Alistipes and Helicobacter shared the dominant proportion of Candidatus _Arthromitus compared with the control chickens. These functional bacteria genera assisted in the transportation and digestion of amino acids, carbohydrates, and ions, synthesis of cellular membranes, and anti-oxidation. Uncultured_organism_g_ Anaerosporobacter, Lactobacillus salivarius, uncultured_bacterium_g_ Ruminococcaceae_UCG-014, uncultured_bacterium_g_ Peptococcus were strongly and positively correlated with body growth performance and anti-oxidation. A metabonomic assay suggested that the secreted of gamma-aminobutyric acid and monobactam was metabolized according to the Kyoto Encyclopedia of Genes and Genomes analysis. In conclusion, Ligilactobacillus salivarius optimized microbial composition of the caecum and secreted functional peptides through gut-brain axis to improve the body growth and antioxidation of broiler chicken.

Goose Hepatic IGFBP2 Is Regulated by Nutritional Status and Participates in Energy Metabolism Mainly through the Cytokine-Cytokine Receptor Pathway.

Changes in the nutritional status of animals significantly affect their health and production performance. However, it is unclear whether insulin-like growth factor-binding protein 2 (IGFBP2) mediates these effects. This study aimed to investigate the impact of changes in nutritional and energy statuses on hepatic IGFBP2 expression and the mechanism through which IGFBP2 plays a mediating role. Therefore, the expression of IGFBP2 was first determined in the livers of fasting/refeeding and overfeeding geese. The data showed that overfeeding inhibited IGFBP2 expression in the liver compared with the control (normal feeding) group, whereas the expression of IGFBP2 in the liver was induced by fasting. Interestingly, the data indicated that insulin inhibited the expression of IGFBP2 in goose primary hepatocytes, suggesting that the changes in IGFBP2 expression in the liver in the abovementioned models may be partially attributed to the blood insulin levels. Furthermore, transcriptome sequencing analysis showed that the overexpression of IGFBP2 in geese primary hepatocytes significantly altered the expression of 337 genes (including 111 up-regulated and 226 down-regulated genes), and these differentially expressed genes were mainly enriched in cytokine-cytokine receptor, immune, and lipid metabolism-related pathways. We selected the most significant pathway, the cytokine-cytokine receptor pathway, and found that the relationship between the expression of these genes and IGFBP2 in goose liver was in line with the findings from the IGFBP2 overexpression assay, i.e., the decreased expression of IGFBP2 was accompanied by the increased expression of LOC106041919, CCL20, LOC106042256, LOC106041041, and IL22RA1 in the overfed versus normally fed geese, and the increased expression of IGFBP2 was accompanied by the decreased expression of these genes in fasting versus normally fed geese, and refeeding prevented or attenuated the effects of fasting. The association between the expression of these genes and IGFBP2 was verified by IGFBP2-siRNA treatment of goose primary hepatocytes, in which IGFBP2 expression was induced by low serum concentrations. In conclusion, this study suggests that IGFBP2 mediates the biological effects induced by changes in nutritional or energy levels, mainly through the cytokine-cytokine receptor pathway.

Female-Biased Expression of R-spondin 1 in Chicken Embryonic Gonads Is Estrogen-Dependent.

The mechanism of sex determination in chickens, especially the molecular mechanism of female ovarian development, has not yet been fully elucidated. Previous studies have shown that RSPO1, which is associated with ovarian development in mammals, might have a conserved role in chickens. In this study, we systematically investigated the spatiotemporal expression pattern of RSPO1 in various tissues, especially gonads, of male and female chicken embryos using qPCR and Western blotting, and we explored its correlation with the expression of key genes in the estrogen pathway using drug treatment or gene overexpression in vivo and in vitro. Our results reveal that RSPO1 was widely expressed in all examined tissues of chicken embryos, showing a female bias in gonadal tissues at both the mRNA and protein levels. Surprisingly, RSPO1 was not differentially expressed between male and female gonadal cells with fadrozole-induced estrogen pathway blockades, and furthermore, estradiol-induced estrogen stimulation altered the expression of RSPO1. In addition, overexpression of RSPO1 in gonadal cells induced the mRNA expression of its downstream target genes, Wnt family member 4 (WNT4) and Catenin beta 1 (CTNNB1), and that of estrogen receptor α (ERα), an estrogen pathway gene. In summary, this study provided new evidence for elucidating the role of RSPO1 in ovarian development in poultry.

Study on the Mechanism of MC5R Participating in Energy Metabolism of Goose Liver.

Nutrition and energy levels have an important impact on animal growth, production performance, disease occurrence and health recovery. Previous studies indicate that melanocortin 5 receptor (MC5R) is mainly involved in the regulations of exocrine gland function, lipid metabolism and immune response in animals. However, it is not clear how MC5R participates in the nutrition and energy metabolism of animals. To address this, the widely used animal models, including the overfeeding model and the fasting/refeeding model, could provide an effective tool. In this study, the expression of MC5R in goose liver was first determined in these models. Goose primary hepatocytes were then treated with nutrition/energy metabolism-related factors (glucose, oleic acid and thyroxine), which is followed by determination of MC5R gene expression. Moreover, MC5R was overexpressed in goose primary hepatocytes, followed by identification of differentially expressed genes (DEGs) and pathways subjected to MC5R regulation by transcriptome analysis. At last, some of the genes potentially regulated by MC5R were also identified in the in vivo and in vitro models, and were used to predict possible regulatory networks with PPI (protein-protein interaction networks) program. The data showed that both overfeeding and refeeding inhibited the expression of MC5R in goose liver, while fasting induced the expression of MC5R. Glucose and oleic acid could induce the expression of MC5R in goose primary hepatocytes, whereas thyroxine could inhibit it. The overexpression of MC5R significantly affected the expression of 1381 genes, and the pathways enriched with the DEGs mainly include oxidative phosphorylation, focal adhesion, ECM-receptor interaction, glutathione metabolism and MAPK signaling pathway. Interestingly, some pathways are related to glycolipid metabolism, including oxidative phosphorylation, pyruvate metabolism, citrate cycle, etc. Using the in vivo and in vitro models, it was demonstrated that the expression of some DEGs, including ACSL1, PSPH, HMGCS1, CPT1A, PACSIN2, IGFBP3, NMRK1, GYS2, ECI2, NDRG1, CDK9, FBXO25, SLC25A25, USP25 and AHCY, was associated with the expression of MC5R, suggesting these genes may mediate the biological role of MC5R in these models. In addition, PPI analysis suggests that the selected downstream genes, including GYS2, ECI2, PSPH, CPT1A, ACSL1, HMGCS1, USP25 and NDRG1, participate in the protein-protein interaction network regulated by MC5R. In conclusion, MC5R may mediate the biological effects caused by changes in nutrition and energy levels in goose hepatocytes through multiple pathways, including glycolipid-metabolism-related pathways.

Preliminary Study on Expression and Function of the Chicken W Chromosome Gene MIER3 in Embryonic Gonads.

The sex chromosomes of birds are designated Z and W. The male is homogamous (ZZ), and the female is heterogamous (ZW). The chicken W chromosome is a degenerate version of the Z chromosome and harbors only 28 protein-coding genes. We studied the expression pattern of the W chromosome gene MIER3 (showing differential expression during gonadogenesis) in chicken embryonic gonads and its potential role in gonadal development. The W copy of MIER3 (MIER3-W) shows a gonad-biased expression in chicken embryonic tissues which was different from its Z copy. The overall expression of MIER3-W and MIER3-Z mRNA and protein is correlated with the gonadal phenotype being higher in female gonads than in male gonads or female-to-male sex-reversed gonads. Chicken MIER3 protein is highly expressed in the nucleus, with relatively lower expression in the cytoplasm. Overexpression of MIER3-W in male gonad cells suggested its effect on the GnRH signaling pathway, cell proliferation, and cell apoptosis. MIER3 expression is associated with the gonadal phenotype. MIER3 may promote female gonadal development by regulating EGR1 and αGSU genes. These findings enrich our knowledge of chicken W chromosome genes and support a more systematic and in-depth understanding of gonadal development in chickens.

The Effects of In Ovo Feeding of Selenized Glucose on Selenium Concentration and Antioxidant Capacity of Breast Muscle in Neonatal Broilers.

This study aims to investigate the impacts of in ovo feeding (IOF) of selenized glucose (SeGlu) on selenium (Se) level and antioxidant capacity of breast muscle in newborn broilers. After candling on 16 day of incubation, a total of 450 eggs were randomly divided into three treatments. On the 17.5th day of incubation, eggs in a control treatment were injected with 0.1 mL of physiological saline (0.75%), while the 2nd group and 3rd group were supplied with 0.1 mL of physiological saline containing 10 µg Se from SeGlu (SeGlu10 group) and 20 µg Se from SeGlu (SeGlu20 group). The results showed that in ovo injection in both SeGlu10 and SeGlu20 increased the Se level and reduced glutathione concentration (GSH) in pectoral muscle of hatchlings (P < 0.05). Compared with the control group, the SeGlu20-treated chicks significantly enhanced the activity of the superoxide dismutase (SOD) and mRNA expression of NAD(P)H quinone dehydrogenase 1 (NQO1) in breast muscle, while there was upregulation in mRNA expressions of glutathione peroxidase 1 (GPX-1) and thioredoxin reductase 1 (TrxR1) and higher total antioxidant capacity (T-AOC) in SeGlu10 treatment (P < 0.05). However, no significant difference on enzyme activities of glutathione peroxidase (GR), glutathione reductase, thioredoxin reductase, concentration of malondialdehyde, and free radical scavenging ability (FRSA) of superoxide radical (O2-•) and hydroxyl radical (OH•) was observed among the three treatments (P > 0.05). Therefore, IOF of SeGlu enhanced Se deposition in breast muscle of neonatal broilers. In addition, in ovo injection of SeGlu could increase the antioxidant capacity of newborn chicks possibly through upregulating the mRNA expression of GPX1, TrxR1, and NQO1, as well as the SOD activity.

Glucose-induced enhanced anti-oxidant activity inhibits apoptosis in goose fatty liver.

The development of mammalian nonalcoholic fatty liver disease is associated with oxidative stress, reduced mitochondrial function, and increased apoptosis in hepatocytes; however, the expressions of mitochondria-related genes are elevated in goose fatty liver, suggesting that there may be a unique protective mechanism in goose fatty liver. The aim of the study was to investigate this protective mechanism in terms of anti-oxidant capacity. Our data showed no substantial differences in the mRNA expression levels of the apoptosis-related genes including B-cell lymphoma-2 (Bcl-2), BCL2-associated X (Bax), cysteinyl aspartate-specific proteinase-3 (Caspase-3), and cysteinyl aspartate-specific proteinase-9 (Caspase-9) in the livers of the control and overfeeding Lander geese groups. The protein expression levels of Caspase-3 and cleaved Caspase-9 were not markedly different between the groups. Compared with the control group, malondialdehyde content was significantly lower (P < 0.01), glutathione peroxidase (GSH-Px) activity, glutathione (GSH) content, and mitochondrial membrane potential levels were higher (P < 0.01) in the overfeeding group. The mRNA expression levels of the anti-oxidant genes superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1), and glutathione peroxidase 2 (GPX2) were increased in goose primary hepatocytes after 40 mM and 60 mM glucose treatment. Reactive oxygen species (ROS) levels were significantly reduced (P < 0.01), whereas the mitochondrial membrane potential was maintained at normal levels. The mRNA expression levels of the apoptosis-related genes Bcl-2, Bax, and Caspase-3 were not substantial. There were no significant differences in the expression levels of Caspase-3 and cleaved Caspase-9 proteins. In conclusion, glucose-induced enhanced anti-oxidant capacity may help protect the function of mitochondria and inhibit the occurrence of apoptosis in goose fatty liver.

No significant pathological symptoms were observed in the liver of goose after overfeeding, suggesting that a specific protection mechanism exists in goose liver. Previous studies have shown that mitochondria may participate in the formation of goose fatty liver by improving its energy metabolism and the production of precursor metabolites. To further understand the role of mitochondria in the formation of goose fatty liver, the present study investigated the changes of mitochondrial function, anti-oxidant capacity, and apoptosis in goose fatty liver. There were found that the level of mitochondrial membrane potential was increased, no apoptosis was observed and anti-oxidant capacity was improved in goose fatty liver, no apoptosis was observed and anti-oxidant genes expressions were increased in goose primary hepatocytes after 40 mM glucose treatment. Our findings imply that apoptosis is inhibited by glucose-induced enhanced anti-oxidant activity in goose fatty liver. Our study not only contributes to revealing the protective mechanism in goose fatty liver but also providing new references for the study of nonalcoholic fatty liver in mammals.

Probiotics-induced Changes in Intestinal Structure and Gut Microbiota Are Associated with Reduced Rate of Pimpled Eggs in the Late Laying Period of Hens.

Production of pimpled or sandpaper-shelled eggs (SE) is a major problem in aged hens. Probiotics can improve eggshell quality; however, the relationship between SE production and gut bacteria remains unclear. Here, 1200 450-d-old Hy-line hens were assigned to four groups (300 hens each), with the control group fed basal diet and treatment groups fed basal diet plus 500, 1000, and 1500 mg/kg of Clostridium butyricum and Bacillus subtilis, respectively. After 4 weeks, probiotics significantly decreased the SE rate from 42.51% to 28.02%. To address why probiotics reduced SE rate, the hens that only produced normal eggs (NE) or SE based on a 2-week assessment were assigned to three groups (NE, SE, and SEP groups; 10 hens each), with the NE and SE groups fed a basal diet and SEP group fed a basal diet plus 1000 mg/kg probiotics. After 4 weeks, ileal tissues from eight birds/group were collected for histomorphological and gene expression analyses, and the ileal content was collected from five birds/group for 16S rDNA sequencing analysis. The data showed that probiotics significantly increased the villus length and ratio of villus length to crypt depth. Quantitative PCR analysis indicated that there were no significant differences in the expression of genes related to tight junctions, nutrient transport, and calcium absorption among the groups (except TRPV6, P<0.001). The 16S rDNA sequencing analysis indicated that the alpha-diversity of gut bacteria in the SEP group was the highest among the groups. The Firmicutes phylum was dominant in the NE and SEP groups, whereas the Proteobacteria phylum was dominant in the SE group. Together, these results suggest that probiotics can significantly influence the intestinal structure and composition of the intestinal microbiota, which may lead to a reduction in the SE rate in aged hens.

The Changes in Microbiotic Composition of Different Intestinal Tracts and the Effects of Supplemented Lactobacillus During the Formation of Goose Fatty Liver.

Intestinal bacteria play an important role in the formation of fatty liver in animals by participating in the digestion and degradation of nutrients, producing various metabolites, and altering the barrier effect of the intestine. However, changes in the gut microbiota during the formation of goose fatty liver are unclear. In this study, 80 healthy Landes geese with similar body weights at 70 days of age were randomly divided into two groups: the control group (n = 48; fed ad libitum) and the overfeeding group (n = 32; overfed). The intestinal contents were collected at 0, 12, and 24 days of overfeeding. The 16S rRNA and metagenomic sequencing analyses showed that the dominant phyla were Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria. At the genus level, Phyllobacterium, Bacteroides, Helicobacter, Lactobacillus, Enterococcus, and Romboutsia were the dominant genera in the goose intestine, and most of them were probiotics. In the control group, the relative abundance of Firmicutes in the jejunum and ileum gradually decreased with time, while that of Proteobacteria increased, whereas in the overfeeding group, the relative abundance of Firmicutes in the jejunum and ileum decreased and then increased with time, while that of Proteobacteria showed an opposite trend. In addition, supplementing Lactobacillus to the diet reduced body weight and fatty liver weight in overfed geese, but increased the weight of abdominal fat, suggesting that Lactobacillus supplementation might affect the transport of nascent fat from the liver to abdominal fat. In conclusion, the species of intestinal-dominant bacteria in the geese are relatively stable, but their relative abundance and function are affected by a number of factors. Overfeeding promotes the metabolism of nutrients in the jejunum and ileum and increases bacterial adaptability to environmental changes by enhancing their ability to process environmental and genetic information more efficiently. These findings suggest that the effect of overfeeding on the composition of intestinal microbiota may indirectly influence the formation of goose fatty liver through the gut/liver axis.

Fasting and Refeeding Affect the Goose Liver Transcriptome Mainly Through the PPAR Signaling Pathway.

Nutrition and energy are essential for poultry growth and production performance. Fasting and refeeding have been widely used to study the effects of nutrition, energy, and related mechanisms in chicken. Previous studies have shown that geese have a strong capacity for fat synthesis and storage; thus, changes in the goose liver transcriptome may be different from those in chicken assessed with a model of fasting and refeeding. However, the responses of the goose liver transcriptome to fasting and refeeding have not yet been addressed. In this study, 36 70-day-old Si Ji geese with similar body weight were randomly assigned to three groups: control (ad libitum feeding), fasting (fasted for 24 h), and refeeding (fast for 24 h followed by 2-h feeding) groups. After treatment, eight geese per group were sacrificed for sample collection. Liver samples from four geese in each group were subjected to transcriptome analysis, followed by validation of differentially expressed genes (DEGs) using quantitative polymerase chain reaction with the remaining samples. As a result, 155 DEGs (73 up-regulated) were identified between the control and fasting groups, and 651 DEGs (321 up-regulated) were identified between the fasting and refeeding groups. The enrichment analyses of Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways showed that fasting mainly influenced material metabolism in the liver, especially lipid metabolism; in contrast, refeeding affected not only lipid metabolism but also glucose and amino acid metabolism. In addition, the peroxisome proliferator-activated receptor (PPAR) signaling pathway may play an important role in lipid metabolism. In conclusion, fasting and refeeding have a strong effect on lipid metabolism in the goose liver; specifically, fasting promotes fatty acid oxidation and inhibits fatty acid synthesis, and refeeding has the opposite effect. The model of fasting and refeeding is suitable for goose nutrition studies.

Screening of MicroRNAs with Potential Systemic Effects Released from Goose Fatty Liver.

Communication between tissues and organs plays an important role in the maintenance of normal physiological functions as well as the occurrence and development of diseases. Communication molecules act as a bridge for interactions between tissues and organs, playing not only a local role in the tissues and organs where they are secreted but also in exerting systemic effects on the whole body via circulation. In this study, blood microRNA-omics analysis of overfed vs. normally fed (control) Landes geese revealed that the content of each of the 21 microRNAs (miRNAs) in the blood of overfed geese was significantly higher than that in the blood of control geese. These miRNAs may have systematic effects in the development of goose fatty liver as well as being candidate markers for the diagnosis of goose fatty liver. We determined the expression of miR-143, miR-455-5p, miR-222a-5p, miR-184, miR-1662, and miR-129-5p using quantitative PCR in goose fatty liver vs. that in normal liver. The expression of these miRNAs, except miR-129-5p, in goose fatty liver was also significantly higher than that in normal liver (P<0.05), suggesting that these blood miRNAs are released from goose fatty liver. In addition, we found that expression of IGFBP5, the predicted target gene of miR-143, was significantly decreased in goose fatty liver vs. the normal liver (P<0.05), indicating that miR-143 may exert both local and systematic effects by inhibiting the expression of IGFBP5, thus promoting the development of goose fatty liver. In conclusion, we identified several miRNAs, including those we validated (i.e., miR-143, miR-455-5p, miR-222a-5p, miR-184, miR-1662, and miR-129-5p) that may serve as candidate markers in the diagnosis of goose fatty liver as well as local and global regulators contributing to the development of goose fatty liver.

Glucose participates in the formation of goose fatty liver by regulating the expression of miRNA-33/CROT.

Glucose oversupply promotes formation of fatty liver, and fatty liver is usually accompanied with hyperglycemia. However, the mechanism by which glucose promotes formation of fatty liver is not very clear. In this study, fatty liver was successfully induced in Landes goose by 19 days of overfeeding with corn-based feed, the overfed geese had a significantly higher level of blood glucose than the normally fed geese (control group). In goose primary liver cells, high level of glucose promoted fat deposition and induced the expression of SREBF2(or SREBP2), a key regulator of lipid metabolism, and its intronic gene, miR-33. Moreover, overexpression of miRNA-33(miR-33) promotes lipid accumulation in goose primary liver cells. Consistently, miR-33 inhibitor suppressed glucose induced lipid accumulation in liver cells. Interestingly, the relative abundance of miR-33 in goose fatty liver was significantly higher than that in normal liver, while the relative mRNA and protein abundances of CROT, the target gene of miR-33, in goose fatty liver were significantly lower than those in goose normal liver. Taken together, these findings suggest that miR-33 mediates glucose promotion of lipid accumulation in goose primary liver cells, and that glucose participates in formation of goose fatty liver by regulating the expression of miR-33/CROT.

Dietary Clostridium butyricum and Bacillus subtilis Promote Goose Growth by Improving Intestinal Structure and Function, Antioxidative Capacity and Microbial Composition.

Probiotics are a substitute for antibiotics in the sense of intestinal health maintenance. Clostridium butyricum and Bacillus subtilis, as probiotic bacteria, have been widely used in animal production. The aim of this study was to investigate the effects of the two probiotic bacteria in geese. A total of 288 1-day old, healthy Yangzhou geese were randomly assigned into 4 groups (A, B, C and D) with 6 replicates of 12 birds each. Group A, as control, was fed a basal diet, and the treatment groups (B, C and D) were fed the basal diet supplemented with 250 mg/kg Clostridium butyricum (the viable count was 3.0 × 106 CFU/g), 250 mg/kg Bacillus subtilis (the viable count was 2.0 × 107 CFU/g), or a combination of the two probiotic bacteria for 70 days, respectively. The results indicated that: compared with the control group, dietary probiotics (1) promoted the growth and feed intake of the geese, (2) increased the absolute weight of duodenum, (3) increased the antioxidative capacity (total antioxidative capacity (T-AOC), total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-PX)) of intestinal mucosa, (4) improved intestinal morphology (the ratio of villus height to crypt depth), (5) but did not induce inflammation and changes of tight junction in the intestine, which was indicated by no induction of pro/inflammatory cytokines (IL-1ß, IL-6, IL-10, TNFAIP3) and tight junction related genes (TJP1 and OCLN). Moreover, dietary probiotics increased the relative abundances of Firmicutes phylum and Lactobacillus genus and decreased the relative abundances of Proteobacteria phylum or Ralstonia genus in the intestinal content. In addition, the alpha diversity (observed species, Chao1, and estimate the number of OTUs in the community(ACE)) was reduced and the predicted functions of intestinal microflora, including peptidases, carbon fixation and metabolic function of starch and sugar, were enhanced by dietary probiotics. In conclusion, dietary probiotics promote the growth of geese by their positive effects on intestinal structure and function, the composition and functions of gut microflora, and intestinal antioxidative capacity.

Uterine structure and function contributes to the formation of the sandpaper-shelled eggs in laying hens.

The avian eggshell is formed in the uterus, and eggshell quality usually decreases markedly in the late phase of hen laying cycles. Production of sandpaper-shelled eggs (SE), a category of eggs with relatively less eggshell quality, causes a great economic loss. Underlying mechanisms of SE formation, however, remain unclear. For the present study, it was hypothesized that alterations in uterine structure and function contribute to SE formation. To test this hypothesis, uterine samples were collected from 450-day-old hens that produced normal eggs (NE) and SE (based on 2-week-long assessments, n = 10) for histomorphological and transcriptome analyses. Compared with the NE group, uteri of the SE group were apparently atrophied. Furthermore, a total of 211 differentially expressed genes (DEGs) were identified in the uteri of hens of the two groups. These DEGs were clustered into 145 gene ontology terms (FDR < 0.05) and enriched in 12 KEGG pathways (P < 0.10), which are primarily related to organ morphogenesis and development, cell growth, differentiation and death, ion transport, endocrine and cell communication, immune response, and corticotropin-releasing hormones. In particular, corticotropin may be an important factor in SE formation because of effects on ion transport. Furthermore, as indicated by lesser abundances of relevant mRNA transcripts, the lesser expression of genes related to ion transport and matrix proteins also contribute to SE production because of effects on eggshell formation. In conclusion, results from this study revealed there were structural and functional differences in the hen uterus in NE and SE groups.

Complement C3 participates in the development of goose fatty liver potentially by regulating the expression of FASN and ETNK1.

Non-alcoholic fatty liver disease (NAFLD) occurs in humans, domestic animals and poultry. Different from upregulation of complement C3 in human NAFLD, C3 expression is inhibited in goose fatty liver (GFL), implying a specific role of C3 in GFL. This study was mainly focused on uncovering the uniqueness of goose liver cells in the regulation of C3 expression and identifying the downstream genes of C3 to improve understanding on the specific role of C3 in GFL. The results showed that C3 expression was inhibited in the liver, muscle and fat tissues of the overfed versus control (normally fed) geese. Oleate and insulin could inhibit C3 expression in goose primary hepatocytes but induce it in mouse primary hepatocytes. A total of 1,123 differentially expressed genes (DEGs) were affected by C3 overexpression and were mainly enriched in immune response/inflammation and catabolism-related KEGG pathways. Additionally, the representative downstream genes (FASN and ETNK1) of C3 could mediate the role of C3 in the development of GFL. In conclusion, the suppression of C3 in GFL is at least partially attributed to hyperinsulinemia, hyperlipidemia and uniqueness of goose liver cells. Complement C3 does not only affect hepatic steatosis but also affect inflammation/immune response in GFL.

Fasting and overfeeding affect the expression of the immunity- or inflammation-related genes in the liver of poultry via endogenous retrovirus.

It is known that nutrition and immunity are connected, but the mechanism is not very clear. Endogenous retroviruses (ERV) account for 8 to 10% of the human and mouse genomes and play an important role in some biological processes of animals. Recent studies indicate that the activation of ERV can affect the expression of the immunity- or inflammation-related genes, and the activities of ERV are subjected to regulation of many factors including nutritional factors. Therefore, we hypothesize that nutritional status can affect the expression of the immunity- or inflammation-related genes via ERV. To verify this hypothesis, the nutritional status of animals was altered by fasting or overfeeding, and the expression of intact ERV (ERVK18P, ERVK25P) and immunity- or inflammation-related genes (DDX41, IFIH1, IFNG, IRF7, STAT3) in the liver was determined by quantitative PCR, followed by overexpressing ERVK25P in goose primary hepatocytes and determining the expression of the immunity- or inflammation-related genes. The data showed that compared with the control group (no fasting), the expression of ERV and the immunity- or inflammation-related genes was increased in the liver of the fasted chickens but decreased in the liver of the fasted geese. Moreover, compared with the control group (routinely fed), the expression of ERV and the immunity- or inflammation-related genes was increased in the liver of the overfed geese. In addition, overexpression of ERVK25P in goose primary hepatocytes can induce the expression of the immunity- or inflammation-related genes. In conclusion, these findings suggest that ERV mediate the effects of fasting and overfeeding on the expression of the immunity- or inflammation-related genes, the mediation varied with poultry species, and ERV and the immunity- or inflammation-related genes may be involved in the development of goose fatty liver. This study provides a potential mechanism for the connection between nutrition and immunity.

Dietary Selenized Glucose Increases Selenium Concentration and Antioxidant Capacity of the Liver, Oviduct, and Spleen in Laying Hens.

Selenized glucose (SeGlu) is a new type of organic selenium (Se) that is synthesized through the selenide reaction of glucose with sodium hydrogen selenide. This study aimed to clarify the influence of dietary SeGlu on the Se level and antioxidant capacity of the liver, oviduct, and spleen in laying hens. A total of 360, 60-week-old, Hy-Line Brown laying hens were randomly assigned to three treatment groups: a basal diet alone (control group, without adding exogenous Se) or the basal diet supplemented with 0.3 mg/kg of Se from sodium selenite (SS) or 5 mg/kg of Se from SeGlu. Diets with SeGlu increased Se levels in the liver, oviduct, and spleen of laying hens (P < 0.001). Compared with the control and SS groups, diet supplemented with SeGlu enhanced glutathione peroxidase (GSH-Px) activity and total antioxidant capacity (T-AOC) in the spleen and oviduct as well as the scavenging ability of 2, 2-diphenyl-1-picrylhydrazyl free radical (DPPH•) in the oviduct (P < 0.05). Compared with the control group, SeGlu treatment resulted in an increase (P < 0.05) in GSH-Px activity, T-AOC, and scavenging abilities of hydroxyl radical and DPPH• in the liver of hens. In addition, dietary SeGlu and SS decreased the hydrogen peroxide level in the oviduct in comparison to the control group (P < 0.05). Therefore, dietary SeGlu increased Se concentration and antioxidant ability in the liver, oviduct, and spleen of laying hens. Moreover, SeGlu may be used as a potential source of Se additive in laying hen production.

GC-TOF-MS-Based Metabolomics Analyses of Liver and Intestinal Contents in the Overfed vs. Normally-Fed Geese.

No overt pathological symptoms are observed in the goose liver with severe steatosis, suggesting that geese may host unique protective mechanisms. Gas chromatography time-of-flight mass spectrometry-based metabolomics analyses of liver and intestinal contents in overfed vs. normally fed geese (26 geese in each treatment) were investigated. We found that overfeeding significantly changed the metabolic profiles of liver and intestinal contents. The differential metabolites mainly belong to fatty acids, amino acids, organic acids, and amines. The differential metabolites were involved in glycolysis/gluconeogenesis, glycerolipid metabolism, the pentose phosphate pathway, fatty acid degradation, the sphingolipid signaling pathway, and the biosynthesis of unsaturated fatty acids. Moreover, we determined the biological effects of arachidonic acid (ARA) and tetrahydrocorticosterone (TD) in goose primary hepatocytes and intestinal cells. Data showed that the mRNA expression of arachidonate 5-lipoxygenase (ALOX5) in goose primary intestinal cells was significantly induced by 0.50 mM ARA treatment. Cytochrome P-450 27A1 (CYP27A1) mRNA expression was significantly inhibited in goose primary hepatocytes by 1 µM TD treatment. In conclusion, the formation of goose fatty liver is accompanied by significant changes in the metabolic profiles of liver and intestinal contents, and the changes are closely related to the metabolisms of glucose and fatty acids, oxidative stress, and inflammatory reactions.

Maintaining intestinal structural integrity is a potential protective mechanism against inflammation in goose fatty liver.

Overfeeding causes severe steatosis but not inflammation in goose liver, suggesting existence of protective components. Previous studies have shown that some intestinal microbes and their metabolites damage intestinal structural integrity and function, thus causing inflammation in the development of human and mouse nonalcoholic fatty liver disease. Therefore, this study hypothesizes that intestinal structural integrity of goose is maintained during overfeeding, which may provide goose fatty liver a protective mechanism against inflammation. To test this hypothesis, 48 seventy-day-old healthy Landes male geese were overfed (as overfeeding group) or normally fed (as control group). Blood and intestine (jejunum, ileum, and cecum) samples were harvested on the 12th and 24th d of overfeeding. Data showed that goose fatty liver was successfully induced by 24 d of overfeeding. Hematoxylin-eosin staining analysis indicated that the arrangement of villi and crypts in the intestine was orderly, and the intestinal structure was intact with no pathological symptoms in the 2 groups. Enzyme-linked immunosorbent assay and quantitative PCR analysis indicated no significant differences in the expression of tight junction and inflammation-related genes as well as plasma lipopolysaccharide concentration between the groups. Ileal hypertrophy and cecal atrophy were observed in the overfed vs. control geese, probably because of change of sphingolipid metabolism. Activation of apoptotic pathway may help cecum avoid necrosis-induced inflammation. In conclusion, healthy and intact intestine provides a layer of protection for goose fatty liver against inflammation. Sphingolipid metabolism may be involved in the adaptation of ileum and cecum to overfeeding. The hypertrophy of ileum makes it an important contributor to the development of goose fatty liver. The atrophy and decline in the function of cecum may be caused by apoptosis induced by overfeeding.