Duck hepatitis B virus infection of Muscovy duck hepatocytes and nature of virus resistance in vivo.

To test the hypothesis that in vivo resistance to hepadnavirus infection was due to resistance of host hepatocytes, we isolated hepatocytes from Muscovy ducklings and chickens, birds that have been shown to be resistant to duck hepatitis B virus (DHBV) infection, and attempted to infect them in vitro with virus from congenitally infected Pekin ducks. Chicken hepatocytes were resistant to infection, but we were able to infect approximately 1% of Muscovy duck hepatocytes in culture. Infection requires prolonged incubation with virus at 37 degrees C. Virus spread occurs in the Muscovy cultures, resulting in 5 to 10% DHBV-infected hepatocytes by 3 weeks after infection. The relatively low rate of accumulation of DHBV DNA in infected Muscovy hepatocyte cultures is most likely due to inefficient spread of virus infection; in the absence of virus spread, the rates of DHBV replication in Pekin and Muscovy hepatocyte cultures are similar. 5-Azacytidine treatment can induce susceptibility to DHBV infection in resistant primary Pekin hepatocytes but appears to have no similar effect in Muscovy cultures. The relatively inefficient infection of Muscovy duck hepatocytes that we have described may account for the absence of a detectable viremia in Muscovy ducklings experimentally infected with DHBV.     

Quantitative real-time PCR primer design, cDNA amplification and sequence analysis from 22 genes mainly associated with lipid metabolism in Pekin (Anas platyrhynchos) and Muscovy (Cairina moschata) ducks

Few genomic tools are available in ducks. To produce some new resources, we have designed Pekin (Anas platyrhynchos) and Muscovy (Cairina moschata) duck-specific primers for 22 genes involved mainly in lipid metabolism, and to a lesser extent in carbohydrate metabolism and other functions. Primers were designed according to duck sequences when available and otherwise from the corresponding conserved regions in chicken and human sequences. These primers allowed quantitative RT-PCR amplification of RNA from Pekin and Muscovy ducks. Amplified cDNA products from both species were sequenced and were found to be very similar to chicken sequences (about 94%). This work provides additional genomic resources and polymorphism information for some genes in duck species and represents a first step towards gene expression analyses in Pekin and Muscovy ducks.     

Sex ratios in mule duck embryos at various stages of incubation

Mule duck hatcheries have long reported varying degrees of unbalance in the sex ratio, with a preponderance of male mules at hatching. The aim of the present study was to assess the distributions of sex ratios at various stages of development in embryos originating from intra- and intergeneric crosses between parental lineages (Muscovy male x Muscovy female, Pekin male x Pekin female, Muscovy male x Pekin female or Mule, and Pekin male x Muscovy female or Hinny). In Experiment I, embryo sexing was performed on Days 1 and 5 of incubation (by multiplex PCR) and at hatching (by vent observation). The sex ratio was not significantly modified during the early stages of embryo development whatever the genetic origin (P>0.05, Days 1 and Day 5) but our results in mule and hinny ducklings confirmed the preponderance of males among normally hatched ducklings originating from the intergeneric lineage (58.9 and 55.4% males in mules and hinnies, respectively; P<0.05 in both cases). Sex ratio (vent sexing) in second grade (cull) ducklings revealed that 68% of these ducklings were females (P<0.05). In Experiment II, the distribution of sex ratio was also performed in mule duck eggs from 6 batches (400,000 eggs/batch) first examined for fertility (candling) on Day 18 of incubation. These results indicate that the percentage of males present in the population of normally hatched ducklings increases when fertility decreases. In addition, this experiment also revealed that 83.7-90.5% of viable male mule embryos develop up to hatching, compared to only 43.0-51.0% of female mule embryos. Given that a deviation in sex ratio during the first stages of incubation is unlikely (Experiment I), it is concluded that the skewed sex ratio of mule ducks at hatching is primarily due to increased late mortality in female mule embryos occurring between egg transfer and hatching. This mortality originated, at least in part, from the intergeneric origin of female mules, and was marked to a greater or lesser extent depending on the initial success of fertilization in a given batch, a possible indication that the initial quality of gametes may selectively exert its influence at the later stages of embryo development.     

Effects of heat stress on antioxidant defense system,inflammatory injury,and heat shock proteins of Muscovy and Pekin ducks: evidence for differential thermal sensitivities

Rising temperatures are severely affecting the mortality, laying performance, and meat quality of duck. Our aim was to investigate the effect of acute heat stress on the expression of heat shock proteins (HSPs: HSP90, 70, 60, 40, and 10) and inflammatory factors (nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2)) and antioxidant enzyme activity (superoxide dismutase (SOD), malondialdehybe (MDA), catalase (CAT), total antioxidant capacity (T-AOC)) in livers of ducks and to compare the thermal tolerance of Pekin and Muscovy ducks exposed to acute heat stress. Ducks were exposed to heat at 39 ± 0.5 °C for 1 h and then returned to 20 °C for 1 h followed by a 3-h recovery period. The liver and other tissues were collected from each individual for analysis. The mRNA levels of HSPs (70, 60, and 40) increased in both species, except for HSP10, which was upregulated in Muscovy ducks and had no difference in Pekin ducks after heat stress. Simultaneously, the mRNA level of HSP90 decreased in the stress group in both species. Morphological analysis indicated that heat stress induced tissue injury in both species, and the liver of Pekin ducks was severely damaged. The activities of several antioxidant enzymes increased in Muscovy duck liver, but decreased in Pekin duck. The mRNA levels of inflammatory factors were increased after heat stress in both duck species. These results suggested that heat stress could influence HSPs, inflammatory factors expression, and the activities of antioxidant enzymes. Moreover, the differential response to heat stress indicated that the Muscovy duck has a better thermal tolerance than does the Pekin duck.     

Is the hepatic metabolism of glucose and linoleic acid influenced by species in overfed ducks?

There are genetic differences in the hepatic glucose and linoleic acid metabolisms between Muscovy and Pekin ducks ad libitum-fed. To understand the effect of overfeeding on the hepatic metabolisms in these two species of ducks, we compared the different pathways of glucose and linoleic acid reaching the liver of Muscovy (Cairina moschata) (n = 6) and Pekin (Anas platyrhynchos) (n = 6) ducks overfed for 1 week and sacrificed 2–4 h after their last meal by using the ex vivo method of liver slices incubated for 16 h with [U-¹⁴C]-glucose, [1-¹⁴C]-linoleic acid and [³⁵S]-methionine added to the survival medium. The glucose was the main precursor of triacylglycerol synthesis in the liver of these two species and its hepatic metabolism was similar between species. The hepatic uptake of linoleic acid was 1.7-fold higher (P = 0.020) in the Muscovy duck than in the Pekin duck leading to a 1.9-fold higher (P = 0.017) esterification of this fatty acid in the liver of the Muscovy duck than in that of the Pekin duck. Finally, both species after 1 week of overfeeding exhibited the same capacity to secrete VLDL remaining insufficient to avoid hepatic steatosis.     

Analysis of the sternotrachealis muscle fibers in some Anseriformes: histochemistry and sex differences

Histochemical characteristics and sizes of the fibers of the sternotrachealis (ST) muscle have been investigated in some Anseriformes (mallard, Pekin duck, Muscovy duck, and goose) of both sexes. A sexual dimorphism has been shown in the muscle of the species examined. In the mallard and Pekin duck, the male ST muscle shows type IIIA fibers in addition to the type I, IIA, and IIB fibers observed also in the female. In the Muscovy duck, the male muscle has only type I and IIA fibers, whereas the female muscle presents type I fibers and both types IIA and IIB fibers. Moreover, the mean frequencies for each fiber type were significantly different between males and females. In the goose, both male and female muscles present only type I and IIA fibers. In all the species examined, the mean areas of each fiber type are significantly different between male and female, being always larger in the male muscles. The anatomical sexual dimorphism observed in the ST muscle is discussed in relation to function.     

Behavioural and physiological fear responses in ducks: genetic cross effects

Mule duck, a cross between a Muscovy drake and a Pekin female, is reported by the farmers to frequently express fear behaviours, such as man avoidance. The genetic basis of fear responses in mule ducks was therefore investigated in this study. According to a previous experiment, the dominant effect of Pekin genotype was hypothesised; however, due to the absence of birds from the reciprocal cross, a superiority of the Pekin in additive effect could not be distinguished from a direct maternal additive effect. In order to clarify this, ducks from the mule genotype, the two parental genotypes (Pekin and Muscovy) and the reciprocal intercross (hinny) underwent a set of physiological and individual behavioural tests of fear. Both parental genotypes were highly fearful but exhibited responses of different patterns: Pekin ducks manifested a higher locomotor activity, whereas the Muscovy ducks showed a higher avoidance to man. Hybrids expressed higher panic responses and specific fear of man than the two parent breeds. Both hybrids expressed similar patterns and the maternal effects were not significant. Significant heterosis effects were found for most of the behavioural responses, in agreement with the fact that higher fear responses were expressed by the hybrids compared to the parental genotypes. A significant heterosis effect was also found for basal adrenal activity; hybrids having higher basal level than parental genotypes. Maximum capacity of adrenal response appeared to be determined by direct additive effects with a superiority of the Pekin genotype.     

Hormonal and metabolic responses to overfeeding in three genotypes of ducks

Muscovy, Pekin and Mule duck are different in their body weight. To make a valid comparison in the lipid metabolism between these three genotypes, overfeeding was carried out by providing the animals with amounts of food in proportion to their body weight. Under these conditions, Muscovy ducks developed a strong liver steatosis, whereas it was not very pronounced in the Mule ducks and even less in the Pekin ducks. On the contrary, Pekin ducks showed a much marked extrahepatic fattening. At the beginning of overfeeding, there was a similarity in the three genotypes as regards the post-heparin lipoprotein-lipase (LPL) activity and the insulin and glucagon concentrations. After 10 days of overfeeding, the LPL activity dramatically fell in Muscovy and in Mule ducks, whereas it remained steady in Pekin ducks. Compared to values found at the beginning of the overfeeding period, plasma glucagon and insulin shown no evolution, except for the insulin of Pekin ducks which was dramatically higher. Those data suggest that high plasma insulin concentrations measured in Pekin ducks after 10 days of overfeeding can be responsible for the maintenance of the LPL activity, which favors the extrahepatic fattening to the detriment of liver steatosis.     

Comparative Proteomic Analysis of the Hepatic Response to Heat Stress in Muscovy and Pekin Ducks: Insight into Thermal Tolerance Related to Energy Metabolism

The Pekin duck, bred from the mallard (Anas platyrhynchos) in china, is one of the most famous meat duck species in the world. However, it is more sensitive to heat stress than Muscovy duck, which is believed to have originated in South America. With temperature raising, mortality, laying performance, and meat quality of the Pekin duck are severely affected. This study aims to uncover the temperature-dependent proteins of two duck species using comparative proteomic approach. Duck was cultured under 39°C ± 0.5°C for 1 h, and then immediately returned to 20°C for a 3 h recovery period, the liver proteins were extracted and electrophoresed in two-dimensional mode. After analysis of gel images, 61 differentially expressed proteins were detected, 54 were clearly identified by MALDI TOF/TOF MS. Of the 54 differentially expressed protein spots identified, 7 were found in both species, whereas 47 were species specific (25 in Muscovy duck and 22 in Pekin duck). As is well known, chaperone proteins, such as heat shock protein (HSP) 70 and HSP10, were abundantly up-regulated in both species in response to heat stress. However, we also found that several proteins, such as α-enolase, and S-adenosylmethionine synthetase, showed different expression patterns in the 2 duck species. The enriched biological processes were grouped into 3 main categories according to gene ontology analysis: cell death and apoptosis (20.93%), amino acid metabolism (13.95%) and oxidation reduction (20.93%). The mRNA levels of several differentially expressed protein were investigated by real-time RT-PCR. To our knowledge, this study is the first to provide insights into the differential expression of proteins following heat stress in ducks and enables better understanding of possible heat stress response mechanisms in animals.     

Susceptibility to duck hepatitis B virus infection is associated with the presence of cell surface receptor sites that efficiently bind viral particles.

To test the hypothesis that susceptibility of hepatocytes to duck hepatitis B virus (DHBV) infection requires cell surface receptors that bind the virus in a specific manner, we developed an assay for the binding of DHBV particles to monolayers of intact cells, using radiolabeled immunoglobulin G specific for DHBV envelope protein. Both noninfectious DHBV surface antigen particles and infectious virions bound to a susceptible fraction (approximately 60%) of Pekin duck hepatocytes. In contrast, binding did not occur to cells that were not susceptible to DHBV infection, including Pekin duck fibroblasts and chicken hepatocytes, and binding to Muscovy duck hepatocytes, which are only weakly susceptible (approximately 1% of cells) to DHBV infection, was virtually undetectable. Within a monolayer, individual Pekin duck hepatocytes appeared to differ markedly in the capacity to bind DHBV, which may explain difficulties that have been encountered in infecting 100% of cells in culture. We have also found that the loss of susceptibility to infection with DHBV that occurs when Pekin duck hepatocytes are maintained for more than a few days in culture correlates with a decline in the number of cells that bind virus particles efficiently. All of these results support the interpretation that the binding event detected by our assay is associated with the interaction between DHBV and specific cell surface receptors that are required for initiation of infection. Our assay may facilitate isolation and identification of hepatocyte receptors for this virus.     

Identification of histone H1.z components in a Muscovy duck (Cairina moschata L.) population

The general patterns of histone H1 proteins from erythrocyte nuclei of Muscovy duck individuals were similar to those of Pekin type ducks both in acetic acid-urea and 2D polyacrylamide gels. We show here that Muscovy duck histone H1.z in the tested population was represented by three different electromorphs, each presumably encoded by a distinct allelic gene. Accordingly, we have identified six phenotypes consisting of the homodimeric and heterodimeric combinations of the three isoforms. The frequency of the presumptive alleles ranged from 0.506 for the main allele z1 to 0.379 for allele z2 and only 0.113 for the rarest allele z3. In addition to a standard set of somatic H1 variants, an unusual protein X, absent in other avian species, was also revealed.     

Does overfeeding enhance genotype effects on energy metabolism and lipid deposition in breast muscle of ducks?

We evaluated the effects of genotype (Muscovy, Pekin and their crossbreed hinny and mule ducks) and feeding levels (overfeeding between 12 and 14 weeks of age vs ad libitum feeding) on energy metabolism and lipid deposition in breast muscle of ducks. Samples of breast muscle (Pectoralis major) were collected at 14 weeks of age from 8 birds per group. Overfeeding induced an accumulation of lipids in breast muscle (1.5- to 1.7-fold, depending on genotype) mainly induced by triglyceride deposition. It also induced a considerable increase in the amounts (expressed as g/100 g of tissue) of saturated and mono-unsaturated fatty acids (SFA, MUFA), while the amounts of poly-unsaturated fatty acids (PUFA) remained unchanged in hinny and Muscovy ducks or slightly increased in Pekin and mule ducks. In breast muscle, overfeeding decreased the activity of the main enzymes involved in lipogenesis from glucose (glucose-6-phosphate dehydrogenase, G6PDH, malic enzyme, ME, acetyl CoA carboxylase, ACX). Lipoprotein lipase (LPL) activity in Pectoralis major muscle was also significantly decreased (-21%). The ability of muscle tissues to catabolize long-chain fatty acids, as assessed by beta-hydroxyacyl CoA dehydrogenase (HAD) activity, was increased in Pectoralis major muscle, as was cytochrome-c oxidase (COX) activity. Hybrid and Pekin ducks exhibited higher levels of ACX and LPL activity in Pectoralis major muscle than Muscovy ducks, suggesting a greater ability to synthesise lipids in situ, and to take up circulating lipids. Total lipid content in breast muscle of hybrid and Pekin ducks was higher than in that of Muscovy ducks. In hybrid and Pekin ducks, lipid composition of breast muscle was characterized by higher amounts of triglycerides, SFA and MUFA than in Muscovy ducks. Finally, oxidative metabolism was greater in Pectoralis major muscles of hybrid and Pekin ducks than in Muscovy ducks, suggesting an adaptative strategy of muscle energy metabolism according to lipid level.     

Influence of lipoprotein-lipase activity on plasma triacylglycerol concentration and lipid storage in three genotypes of ducks.

The lipoprotein-lipase (LPL) hydrolyses the triacylglycerols (TG) secreted by the liver and, thus, allows the storage of lipids onto the extrahepatic tissues. The LPL activity has been studied by injection of LPL antibodies in three genotypes of ducks (Muscovy (Cairina moschat), Pekin (Anas plathyrhynchos) and Mule (hybrids of male Muscovy ducks and female Pekin ducks)) under overfeeding condition. The results show a similar weight gain between injected and control animals. A higher liver steatosis is observed in Mule ducks (616+/-18 g; 8.79% of body mass (BW)) and Muscovy ducks (514+/-13 g; 7.05% BW) compared to Pekin ducks (353+/-21 g; 5.89% BW, p<0.05). Pekin ducks showed a much marked extrahepatic fattening of abdominal and subcutaneous adipose tissues. The LPL activity was evaluated by comparing the evolution of the plasma TG concentrations after injections of saline (control animals) or injections of specific LPL-antibodies. Inhibition of LPL activity performed by intravenous injections of LPL-antibodies showed a spectacular increase in the plasma TG concentrations in the three genotypes. That increase was considerably higher in Pekin ducks (98+/-10 g/L) compared to Muscovy ducks (35+/-2 g/L, p<0.01) and Mule ducks (30+/-4 g/L, p<0.01). Those data suggest that a high export of lipids synthesized in liver and a high LPL activity occur in overfed Pekin ducks, which can favour the extrahepatic fattening to the detriment of the liver steatosis, and conversely in overfed Muscovy and Mule ducks.     

Is ability to hepatic steatosis influenced by age at the beginning of the overfeeding period in Muscovy and Pekin ducks?

The aim of this study was to analyse the effects of species (Muscovy and Pekin ducks) and age at the beginning of the overfeeding period on fatty liver production, carcass composition and lipid and moisture content of the liver and breast muscle. We reared four groups of 40 ducks per species for the study, starting at 2-week intervals in order to have four different ages together at the beginning of the overfeeding period (10, 12, 14 and 16 weeks). At the end of the overfeeding period, all ducks were slaughtered. Our results confirmed the high levels of difference in carcass composition and lipid content in the plasma, liver and breast muscle between Muscovy and Pekin ducks at all ages. Pekin ducks were not able to develop a high degree of hepatic steatosis, but had increased lipid storage in peripheral adipose and muscle tissues than Muscovy ducks. However, the fatty liver weight of Pekin ducks increased with age, with lipid deposition in the liver and peripheral tissues. The ability of Muscovy ducks to produce fatty livers remained unchanged with age in line, with lipid deposition in the liver and peripheral tissues. The sites of lipid deposition thus depend on species and not on the physiological maturity of ducks.     

Chromosome-level genome assembly of the Muscovy duck provides insight into fatty liver susceptibility

The Muscovy duck (Cairina moschata) is an economically important poultry species, which is susceptible to fatty liver. Thus, the Muscovy duck may serve as an excellent candidate animal model of non-alcoholic fatty liver disease. However, the mechanisms underlying fatty liver development in this species are poorly understood. In this study, we report a chromosome-level genome assembly of the Muscovy duck, with a contig N50 of 11.8 Mb and scaffold N50 of 83.16 Mb. The susceptibility of Muscovy duck to fatty liver was mainly attributed to weak lipid catabolism capabilities (fatty acid β-oxidation and lipolysis). Furthermore, conserved noncoding elements (CNEs) showing accelerated evolution contributed to fatty liver formation by down-regulating the expression of genes involved in hepatic lipid catabolism. We propose that the susceptibility of Muscovy duck to fatty liver is an evolutionary by-product. In conclusion, this study revealed the potential mechanisms underlying the susceptibility of Muscovy duck to fatty liver.     

Does overfeeding enhance genotype effects on liver ability for lipogenesis and lipid secretion in ducks?

We evaluated the effects of genotype (Muscovy, Pekin and their crossbreed hinny and mule ducks) and feeding levels (overfeeding between 12 and 14 weeks of age vs ad libitum feeding) on liver ability for lipogenesis and lipid secretion in ducks. Samples of liver and blood were collected at 14 weeks of age from 8 birds per group. Plasma levels of insulin was considerably increased in overfed ducks (1.9-fold), stimulating the hepatic activity of the main enzymes involved in lipogenesis from glucose (glucokinase, GK, glucose-6-phosphate dehydrogenase, G6PDH, malic enzyme, ME, acetyl CoA carboxylase, ACX), while cytochrome-c oxidase (COX) activity, indicating overall oxidation ability of energy-yielding substrates, remained unchanged. Plasma levels of triglycerides, phospholipids and total cholesterol were therefore increased (1.9, 3.7, 1.6 and 1.6-fold, respectively). Glycaemia also significantly increased (+8%). Pekin ducks exhibited higher levels of GK and G6PDH activity in the liver than Muscovy ducks, suggesting a greater ability to use glucose consistent with their lower glycaemia. Muscovy ducks had greater ACX activity, suggesting greater ability to synthesise lipids. However, plasma lipid levels were much higher in Pekin ducks than in Muscovy ducks, suggesting a greater ability to export lipids from the liver. Values for the different criteria measured in this study were intermediate or similar in hinny and mule ducks to those of parental species. The high values for GK, G6PDH, ME and ACX activity in hybrid ducks enabled them to produce heavy fatty livers with the same chemical and lipid composition as Muscovy ducks and characterised by high amounts of triglycerides (around 96% of total lipids), and saturated and mono-unsaturated fatty acids.     

Insulin effect on lipogenesis and fat distribution in three genotypes of ducks during overfeeding

In waterfowl, the response to overfeeding differs from one genotype to the other. Pekin ducks generally store lipids in the peripheral tissues while Muscovy and mule ducks promote hepatic lipid storage. A possible reason for these various susceptibilities to hepatic steatosis could be a difference in insulin sensitivity. We suggest a resistance to insulin in Pekin ducks. In the present work we investigate the action of insulin on glucose and lipid metabolisms for the three overfed genotypes. Regardless of the kind of genotype, all ducks appear to be sensitive to insulin: their glycemia is lower when the animals are treated with insulin. Insulin-treated Muscovy and Pekin ducks present a lower increase in total body weight (? 16.5% for Muscovy; ? 8.3% for Pekin); and a significantly lower liver weight than the controls (? 9.6% and ? 18.3%). The percentage of total lipids in the liver is higher in the controls than in the insulin-treated Pekin and mule ducks (respectively ? 40.4% and ? 34.7%), which means a decreased hepatic lipogenesis. Pekin ducks present a higher pectoral muscle weight when the individuals are insulin-treated (+ 9.7%). Lipoprotein lipase (LPL) activity appears to be significantly higher in insulin-treated Pekin and Muscovy ducks (1.39 and 3.38 times greater than controls). Insulin-treated mule ducks present a decrease of muscle and abdominal lipid storage compared to controls (? 11.6% and ? 13.8%). In this experiment, exogenous insulin has induced an increase of lipid oxidation and has led to a less favorable use and storage of dietary glucose. The hypothesis of insulin-resistance of Pekin ducks is not verified.     

Epidemiological investigation and genome analysis of duck circovirus in Southern China

Duck circovirus (DuCV), a potential immunosuppressive virus, was investigated in Southern China from March 2006 to December 2009 by using a polymerase chain reaction (PCR) based method. In this study, a total of 138 sick or dead duck samples from 18 different farms were examined with an average DuCV infection rate of ∼35%. It was found that ducks between the ages of 40∼60 days were more susceptible to DuCV. There was no evidence showing that the DuCV virus was capable of vertical transmission. Farms with positive PCR results exhibited no regularly apparent clinical abnormalities such as feathering disorders, growth retardation or lower-than-average weight. The complete genomes of 9 strains from Fujian Province and 1 from Zhejiang Province were sequenced and analyzed. The 10 DuCV genomes, compared with others genomes downloaded from GenBank, ranged in size from 1988 to 1996 base pairs, with sequence identities ranging from 83.2% to 99.8%. Phylogenetic analysis based on genome sequences demonstrated that DuCVs can be divided into two distinct genetic genotypes, Group I (the Euro-USA lineage) and Group II (the Taiwan lineage), with approximately 10.0% genetic difference between the two types. Molecular epidemiological data suggest there is no obvious difference among DuCV strains isolated from different geographic locations or different species, including Duck, Muscovy duck, Mule duck, Cheery duck, Mulard duck and Pekin duck.