Identification of key genes and pathways in duck fatty liver syndrome using gene set enrichment analysis.

High-laying ducks are often fed high-energy, nutritious feeds to maintain high productivity, which predisposes them to lipid metabolism disorders and the development of fatty liver syndrome (FLS), which seriously affects production performance and has a substantial economic impact on the poultry industry. Therefore, it is necessary to elucidate the mechanisms underlying the development of fatty liver syndrome. In this study, seven Shan Partridge ducks, each with fatty liver syndrome and normal laying ducks, were selected, and Hematoxylin Eosin staining (HE staining), Masson staining, and transcriptome sequencing were performed on liver tissue. In addition to exploring key genes and pathways using conventional analysis methods, we constructed the first Kyoto Encyclopedia of Genes and Genomes (KEGG) database-based predefined gene set containing 12,764 pathways and 16,836 genes and further performed gene set enrichment analysis (GSEA) on the liver transcriptome data. Finally, key nodes and biological processes were identified via the protein-protein interaction (PPI) network. The results showed that the liver in the FL group exhibited steatosis and fibrosis, and a total of 3,663 genes with upregulated expression versus 2,296 downregulated genes were screened by conventional analysis. GSEA analysis and PPI network analysis revealed that the liver in the FL group exhibited disruption of the mitochondrial electron transport chain, leading to decreased oxidative phosphorylation and the secretion of excessive proinflammatory factors amid the continuous accumulation of lipids. Under continuous chronic inflammation, cell cycle arrest triggers apoptosis, while fibrosis becomes more severe, and procarcinogenic genes are activated, leading to the continuous development and deterioration of the liver. In conclusion, the predefined gene set constructed in this study can be used for GSEA, and the identified hub genes provide useful reference data and a solid foundation for the study of the genetic regulatory mechanism of fatty liver syndrome in ducks.

The impact of Bacillus subtilis DSM32315 and L-Threonine supplementation on the amino acid composition of eggs and early post-hatch performance of ducklings.

Poultry requires Threonine, an essential amino acid, and its metabolites for proper metabolic function. Threonine is crucial in the biosynthesis of mucin, which is essential for intestinal health and nutrient absorption. Bacillus subtilis (B. subtilis) is a potential substitute for antibiotic growth promoters in the poultry industry. The current study was designed to evaluate the simultaneous effect of L-Threonine (Thr) and B. subtilis DSM32315 supplementation on laying duck breeders in order to maximize performance. A total number of 648 female 23-week-old Longyan duck breeders were assigned to a 3 × 2 factorial design with six replicates of 18 birds per replicate. L-Thr was added to the control diet at concentrations of 0, 0.7, and 1.4 g/kg, equating to 3.9, 4.6, and 5.3 g Thr/kg, with or without B. subtilis strain DSM 32315 (0.0 and 0.5 g/kg). Increasing Thr concentrations improved egg production and ducklings' hatchling weight (p < 0.05). In addition, L-Thr supplementation resulted in a tendency for decreased feed conversion ratio without affecting egg quality. There was no significant effect (p > 0.05) of the dietary Thr levels on egg yolk and albumen amino acid concentrations. In contrast, the addition of B. subtilis decreased the concentrations of amino acids, excluding proline, in the egg white (albumen) and the egg yolk (p < 0.05). Furthermore, the supplementation of B. subtilis decreased (p < 0 0.001) the hatching weight of ducklings. The addition of B. subtilis without L-Thr decreased (p < 0.05) the hatchability of fertile eggs and the hatching weight of ducklings compared to those of ducks fed dietary L-Thr along with B. subtilis (p < 0.001). The combining L-Thr at 0.7 g/kg with B. subtilis DSM 32315 at 0.5 g/kg could increase eggshell quality, hatchability, and hatching weight. The current study revealed that the combination supplemented of L-Thr and B. subtilis DSM 32315 is recommended due to its positive effects on the eggshell percentage, hatchability and the body weights of newly hatched ducklings when dietary Thr was added at a rate of 0.7 g/kg and B. subtilis DSM 32315 at 0.5 g/kg. In addition, adding L-Thr separately at 0.7 g/kg could improve the egg production of duck breeders. Further studies are required to find the proper dosages of B. subtilis DSM 32315 with co-dietary inclusion of limiting amino acids in the diets of duck breeders. The findings of these trials will support feed additive interventions to transition into antibiotic-free diets.

Dietary supplementation with 2-hydroxy-4-methyl(thio) butanoic acid and DL-methionine improves productive performance, egg quality and redox status of commercial laying ducks.

This experiment aimed to study the effects of supplemental methionine sources, 2-hydroxy-4 methyl(thio) butanoic acid (HMTBa) and DL-Methionine (DL-Met), on productive performance, egg quality, and redox status of laying ducks. A total of 792 healthy 25-wk-old Longyan laying ducks with similar body weights were randomly allotted to 11 treatment groups. Each treatment group had 6 replicates of 12 ducks. The trial lasted for 16 wk. Ducks were fed a basal deficient diet (Met: 0.24%; Met + Cys: 0.51%) or supplemented with DL-Met or HMTBa at 0.05%, 0.12%, 0.19%, 0.26%, and 0.33% of diet, respectively. Compared with the basal diet, supplementation with either DL-Met or HMTBa increased the average egg weight, egg mass, and decreased feed to egg ratio during the whole trial period (P < 0.05). Albumen weight and its ratio to total egg weight were increased, but yolk and shell ratio, albumen height, Haugh unit and shell breaking strength were decreased (P < 0.05). Dietary DL-Met or HMTBa supplementation increased taurine, methionine, leucine, tryptophan and arginine content, and decreased serine and lysine content in plasma (P < 0.05). The redox status of laying ducks was improved by enhancing the glutathione peroxidase and catalase activities, glutathione content and its ratio relative to glutathione (oxidized) content and decreasing malondialdehyde content and increasing mRNA expression of superoxide dismutase-1, glutathione peroxidase-1, hemeoxygenase-1 and nuclear factor-like 2 in liver and ileum with the supplementation of DL-Met or HMTBa (P < 0.05). Liver health status measured by average area proportion lipid droplet was improved with supplementation of DL-Met or HMTBa (P < 0.05). Villus height and villus height to crypt depth ratio in the ileum and the ileal gene expression of tight junction protein and occludin were increased with DL-Met or HMTBa supplementation (P < 0.05). Taken together, these results suggested that the efficacy of dietary supplementation of HMTBa was similar to DL-Met, and it ranged from 98% to 100% for productive performance and egg albumen ratio in laying ducks (25 to 41 wk).

Age-related changes in eggshell physical properties, ultrastructure, calcium metabolism-related serum indices, and gene expression in eggshell gland during eggshell formation in commercial laying ducks.

This study evaluated the changes in eggshell mechanical properties, ultrastructure, calcium metabolism-related serum indices, and gene expression in eggshell gland during eggshell formation between laying ducks in the peak (young duck) and late phase (aged duck) of production. A total of 84 healthy young (31 wk of age) and 84 healthy aged (65 wk of age) Longyan laying ducks were each divided into 6 replicates of 14 birds, and caged individually. All the ducks were fed in one house with the same corn-soybean meal-based diet for 5 wk. The eggshell mechanical properties (shell proportion, thickness, breaking strength, and fracture toughness) and chemical components (matrix proteins, calcium, phosphorus, and magnesium) decreased in aged laying ducks (P < 0.05). Shell structural indices: total thickness, effective thickness and its proportion decreased, whereas mammillary knob width and its proportion increased (P < 0.05). The regulation values of early fusion, cuffing, caps, and total score of mammillary knobs were higher in aged laying ducks relative to the young ducks (P < 0.05). During the initial, growth and terminal stages of eggshell formation, shell thickness and breaking strength (terminal), shell weight, and its proportion (terminal) decreased in aged laying ducks (P < 0.05). Ultrastructural changes during shell formation indicated that the mammillary-knob density and effective thickness decreased (P < 0.05). Decreases occurred in serum content of phosphorus (growth), and estradiol and calcium contents (terminal) (P < 0.05). Relative expression of Ca2+ transporter and HCO3- exchanger, and matrix proteins genes decreased in aged laying ducks (P < 0.05) at all stages of eggshell formation. Collectively, the decreased incidence of early fusion and caps, increased thickness and width of mammillary knobs, and decreased effective thickness are the crucial differences leading to the compromised mechanical properties of eggshell in the late laying period. A disturbed regulation of calcium metabolism and uterine expression of ion transporters, especially for HCO3- exchange of aged laying ducks likely contribute to age-induced ultrastructural deterioration of the eggshell.

Dietary zinc supplementation affects eggshell quality and ultrastructure in commercial laying ducks by influencing calcium metabolism.

This study evaluated dietary Zn supplementation on productive performance, eggshell quality and ultrastructure, and calcium metabolism during eggshell formation in laying ducks. A total of 360 Longyan laying ducks (45-wk) were randomly divided into 5 treatment groups with 6 replicates of 12 birds each and fed for 20 wk. The 6 treatments fed the basal diet supplemented with 0 (control), 20, 40, 80, or 160 mg Zn/kg (ZnSO4·H2O). Dietary supplemental level at 80 mg/kg increased egg production (4.3%) and mass (5.7%), and decreased FCR (2.9%) compared to the basal diet, and these indices increased quadratically with increasing Zn supplemental levels (P < 0.05). The shell breaking strength (15.8%) and fracture toughness (10.6%) were higher with the supplementation of Zn at 80 mg/kg than the basal diet, and increased quadratically with Zn supplementation (P < 0.05). Dietary supplementation of Zn at 80 mg/kg improved shell ultrastructure by increasing total (9.0%) and effective thickness (14.2%) and decreasing mammillary thickness (12.0%), and their responses were quadratic with increasing Zn levels (P < 0.05). The supplementation of Zn affected the calcium contents in plasma, tibias and ulna, ulna phosphorus content, and linear and quadratic effects were observed, and higher values were observed with 160 mg/kg Zn supplementation than control (P < 0.05). The supplemental Zn level at 80 mg/kg increased shell effective thickness in growth stage (P < 0.05), and shell calcium and phosphorus content in initial and growth stages (P < 0.05). Dietary Zn supplementation did not affect the gene expression of Ca2+ transporters in the eggshell gland, but affected the expression of HCO3- exchanger in initial and growth stage (P < 0.05). Overall, dietary Zn supplementation could improve productive performance and shell quality in laying ducks at late phase of production, and calcium metabolism and deposition were modulated by Zn influencing HCO3- secretion and thus affecting shell ultrastructure and quality. A supplemental level of 80 mg/kg Zn in the diet with a basal content of 34.0 mg/kg was optimal, and higher level (160 mg/kg) decreased shell calcium deposition by depressing its metabolism.

Effects of dietary barley inclusion and glucanase supplementation on the production performance, egg quality and digestive functions in laying ducks.

This study evaluated the effects of barley inclusion and glucanase supplementation on the productive performance and digestive function in laying ducks. The experiment used a randomized design with a 5 × 2 factorial arrangement of 5 graded levels of barley (0%, 15%, 30%, 45% and 60%) with or without 1.5 g/kg ß-1,3-1,4-glucanase (15,000 U/kg). During the experimental period of 120 d, the weight and total number of eggs within each pen were recorded daily, and egg quality was determined every 4 wk. At the end of the experiment, 3 randomly selected ducks within each replicate were sacrificed, then duodenal digesta and jejunal mucosa was collected. Dietary inclusion of barley had no effects on egg production, daily egg mass or FCR, but supplementation with glucanase improved egg production and FCR (P < 0.01). Barley did not affect feed intake of laying ducks, but glucanase tended to increase feed intake (P = 0.09). Neither barley nor ß-glucanase had effects on the egg quality variables, except for yolk color score, which was decreased with increasing barley supplementation. Glucanase, but not barley, increased the activity of chymotrypsin and amylase in duodenal digesta. Barley inclusion affected the activity of alkaline phosphatase and maltase in jejunal mucosa (P < 0.05), but ß-glucanase had no effects on the activity of these brush border enzymes. Barley inclusion increased the glucan content in duodenal digesta, but supplementation of glucanase to barley-based diet reduced digesta glucan content and reduced total volatile fatty acids and increased the proportion of acetic acid in cecal contents. The results indicate that, without glucanase, the optimal dietary barley level in the diets of laying ducks is about 13% for maximal production performance; glucanase supplementation of the barley diets improved production performance, probably through enhancing digestive function.

Tryptophan in poultry nutrition: Impacts and mechanisms of action.

Many studies have shown that productivity, immune system, antioxidant status, and meat and egg quality can be optimized by dietary supplementation with amino acids that are not usually added to poultry diets. Understanding the effects of these amino acids may encourage feed manufacturers and poultry producers to include them as additives. One of these amino acids is tryptophan (Trp). The importance of Trp is directly related to its role in protein anabolism and indirectly related to its metabolites such as serotonin and melatonin. Thus, Trp could affect the secretion of hormones, development of immune organs, meat and egg production, and meat and egg quality in poultry raised under controlled or stressed conditions. Therefore, this review discusses the main roles of Trp in poultry production and its mode (s) of action in order to help poultry producers decide whether they need to add Trp to poultry diets. Further areas of research are also identified to address information gaps.

Nutritional modulation of fertility in male poultry.

The increased consumption of protein derived from poultry demands greater poultry production, but increased poultry production (meat and eggs) is dependent on the fertility of the parent flocks. Clearly, the fertility of poultry flocks is associated with the fertility of both males and females, but the low numbers of males used for natural or artificial insemination mean that their role is more important. Thus, enhancing the semen volume, sperm concentration, viability, forward motility, and polyunsaturated fatty acids in sperm, as well as protecting against oxidative damage, could help to optimize the sperm membrane functionality, mitochondrial activity, and sperm-egg penetration, and thus fertility. Therefore, this review summarizes the nutritional factors that could improve the fertility of poultry males as well as their associated mechanisms to allow poultry producers to overcome low-fertility problems, especially in aging poultry males, thereby obtaining beneficial impacts on the poultry production industry.

Effects of constant or intermittent high temperature on egg production, feed intake, and hypothalamic expression of antioxidant and pro-oxidant enzymes genes in laying ducks.

Heat stress is a major environmental factor contributing to lower production of poultry. The objective of present study was to evaluate the influence of constant or intermittent high temperature on the production performance and redox status of plasma and hypothalamus in laying ducks. A total of 288 weight- and laying-matched laying ducks were randomly assigned to 1 of 4 treatments (each with 6 replicates of 12 birds): control, pair-fed, constant high temperature (24 h, 34 ± 1°C), and intermittent high temperature (10 h, 34 ± 1°C). Blood and hypothalamic tissue samples were collected on days 1, 21, and 55 to determine redox status. Average daily feed intake and egg weight was reduced (P < 0.001) during imposition of both high-temperature treatments but was not different (P > 0.05) among the treatments during the recovery period. Lower (P < 0.05) egg mass was observed in pair-fed and intermittent high-temperature treatment during high-temperature period and in constant high temperature during the recovery period. Haugh units from high temperature-treated ducks were significantly lower than those from control or pair-fed ducks (P < 0.05) during the high-temperature period. Both models of heat exposure decreased plasma concentrations of glutathione (GSH) at day 1, and constant high temperature decreased plasma activity of GSH peroxidase (GSH-PX) at day 21 (P < 0.05). Hypothalamic expression of antioxidant genes GSH reductase (GR) and mitochondrial NADH dehydrogenase subunit (Complex Ι) were decreased by both high-temperature treatments at day 1. Hypothalamic expression of genes for pro-oxidant enzymes cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), and cytochrome P450 7A1 (CYP7A1) were decreased (P < 0.05) by both models of high temperature but transcripts of cyclooxygenase-1 (COX-1) of ducks that were pair-fed or were exposed to constant high temperature were increased at day 21. The transcripts of NADPH oxidase 1 (NOX-1) were decreased at day 1 by both high-temperature treatments (P < 0.05) but increased during the recovery period. These results indicate that, for laying ducks, intermittent high temperature caused much greater negative production performance effects than constant high temperature during high-temperature period, but laying ducks exposed to constant high temperature tend to take longer to recover their production performance. High-temperature stress, either constant or intermittent, altered hypothalamic expression of antioxidation and pro-oxidation genes.

Nutritional requirements of meat-type and egg-type ducks: what do we know?

The demand for duck meat, duck eggs, and associated products is increasing each year. Classic and modern selection programs have been applied to enhance the economic traits of ducks to satisfy the requirements of consumers and enhance the incomes of producers. The nutritional requirements of unselected ducks may not be adequate, however, to fulfill the potential productivity performance of modern birds, including both meat-type and egg-type ducks. In particular, an imbalanced diet is associated with low productive performance and signs of nutritional deficiency (if insufficient nutrients are supplied), as well as with high feed costs and manure problems that reflect flock health and welfare (if excessive nutrients are supplied). Thus, the main aim of this review is to summarize the results of previous studies that estimated the nutrient requirements of meat-type and egg-type ducks in order to evaluate current knowledge and to identify further issues that need to be addressed. In addition, the results obtained in previous studies are compared in order to understand how to lower commercial feed costs, fulfill the genetic potential of selected ducks, protect the environment from pollution, and satisfy the welfare and health needs of ducks.

Effects of dietary methionine on productivity, reproductive performance, antioxidant capacity, ovalbumin and antioxidant-related gene expression in laying duck breeders.

The study investigated whether dietary methionine (Met) affects egg weight and antioxidant status through regulating gene expression of ovalbumin (OVAL), nuclear factor erythroid 2 like 2 (Nrf2) and haem oxygenase 1 (HO-1) in laying duck breeders. Longyan duck breeders (n 540, 19 weeks) were randomly assigned to six treatments with six replicates of fifteen birds each. Breeders were fed diets with six Met levels (2·00, 2·75, 3·50, 4·25, 5·00 and 5·75 g/kg) for 24 weeks. The egg weight (g), egg mass (g/d), feed conversion ratio, hatchability, 1-d duckling weight, albumen weight, albumen proportion and OVAL mRNA level improved with dietary Met levels, whereas yolk proportion decreased (P<0·05). The weight of total large yellow follicles increased linearly (P<0·001) and quadratically (P<0·05) with dietary Met concentration, and their weight relative to ovarian weight showed a linear (P<0·05) effect. Dietary Met level had a linear (P<0·05) and quadratic (P<0·001) effect on the gene expression of glutathione peroxidase (GPX1), HO-1 and Nrf2, and quadratically (P<0·05) increased contents of GPX and total antioxidant capacity (T-AOC) in liver of duck breeders. In addition, maternal dietary Met enhanced gene expression of GPX1, HO-1 and Nrf2, increased contents of GPX and T-AOC and reduced carbonylated protein in the brains of hatchlings. Overall, dietary Met concentration affected egg weight and albumen weight in laying duck breeders, which was partly due to gene expression of OVAL in oviduct magnum. A diet containing 4·0 g Met/kg would achieve optimal hepatic GPX1 and Nrf2 expression, maximise the activity of GPX and minimise lipid peroxidation.

Estimation of dietary arginine requirements for Longyan laying ducks.

This study aimed to establish the arginine requirements of Longyan ducks from 17 to 31 wk of age based on egg production, egg quality, plasma, and ovarian indices, as well as the expression of vitellogenesis-related genes. In total, 660 Longyan ducks with similar body weight at 15 wk of age were assigned randomly to 5 treatments, each with 6 replicates of 22 birds, and fed a corn-corn gluten meal basal diet (0.66% arginine) supplemented with either 0, 0.20%, 0.40%, 0.60%, or 0.80% arginine. Dietary arginine did not affect egg production by laying ducks, but it increased (linear, P < 0.01) the egg weight at 22 to 31 and 17 to 31 wk of age. Dietary arginine increased the yolk color score (linearly, P < 0.05) and the yolk percentage (quadratic, P < 0.05), where the maximum values were obtained with 1.26% arginine. Dietary arginine affected the total shell percentage and shell thickness, with the highest values using 1.46% arginine (P < 0.01). The weight and number of small yellow follicles (SYFs) increased (quadratic, P < 0.05) with the dietary arginine level and there was a quadratic response (P < 0.05) in terms of the SYFs weight/ovarian weight; the highest values were obtained in ducks fed 1.26% arginine. The plasma arginine concentration exhibited a quadratic (P < 0.05) response to dietary arginine. The plasma progesterone concentration decreased (linear, P < 0.05) as dietary arginine increased. The mRNA abundance of the very low density lipoprotein receptor-b increased in the second large yellow follicle membranes (quadratic, P < 0.05) with the dietary arginine level, where the highest value occurred with 1.26% arginine. According to the regression model, the dietary arginine requirements for Longyan laying ducks aged 17 to 31 wk are 1.06%, 1.13%, 1.22%, and 1.11% to obtain the maximum yolk percentage, SYFs number, SYFs weight, and SYFs weight/ovarian weight, respectively.

Purification and partial characterization of an acidic α-amylase from a newly isolated Bacillus subtilis ZJ-1 that may be applied to feed enzyme.

An amylase-producing strain was isolated from soy sauce and designated as Bacillus subtilis ZJ-1. Purification of α-amylase from B. subtilis ZJ-1 to homogeneity by ethanol fractionation, ultrafiltration, and Sephadex G-100 gel filtration resulted in recovery of 8.9% and a specific activity of 542.7 U/mg protein. The molecular mass was estimated to be 58 kD by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme reached its maximum activity at a pH of 5.0 and a temperature of 50°C. The enzyme remained at 89.4 ± 3.0% of its activity at 40°C. The enzyme retained 87.7 ± 3.7% and 63.4 ± 2.9% of its original activity at 40°C after a 60-min incubation in the presence of 5 mM CaCl2 at a pH of 5.0 and 4.0, respectively. These properties indicate that the novel enzyme has a theoretically high survival rate and excellent starch catalytic efficiency in the typical chicken gastrointestinal-tract environment (pH 3.5-7.0, 40°C). In addition, the enzyme remained at 78.4 ± 3.6% of its activity after a 5-min incubation at 80°C, which demonstrates that the enzyme could maintain a high survival rate in the pelleting process of feed production. The characteristics just described make this enzyme a good candidate for use as a chicken feed enzyme.