Optimization of embryonic thermal programming confirms increased liver fattening in mule ducks and changes in lipid metabolism.

Introduction: The embryonic thermal programming (TM) in birds has been shown to impact several physiological parameters such as resistance to thermal stress, muscle growth or immunity. In mule ducks, it has recently been shown that TM can induce metabolic programming resulting in increased liver weight and fat storage after overfeeding. However, a decrease in hatchability and foie gras quality was also observed, suggesting that this technique needs to be optimized. Here, we tested a new thermal manipulation condition determined with the objective of avoiding negative impacts while maintaining or improving liver properties. Methods: The eggs of the control group were incubated at 37.6°C during the whole incubation period while those of the experimental group (TM group) were incubated at 39.3°C 16 h/24 h from the 11th day of incubation to the 21st. After hatching, all the animals were fed and raised under the same conditions until the age of 12 weeks. At this stage, one part of the animals was overfed and then slaughtered 2 h (to measure rapid changes in metabolism) or 10 h after the last meal (to obtain the best technological yields), while the other part was ration-fed and slaughtered 2 h after the last meal, at the same age. Results: An 8% increase in foie gras production was measured in the TM group compared to the control group without altering the quality of the final product (nor hatchability), confirming the successful optimization of the metabolic programming. Interestingly, these results allowed us not to reject the previously suggested hypothesis of a potential delay in metabolic processes involved in liver fattening in programmed animals, in particular by measuring a trend reversal regarding the amount of total hepatic lipids in both groups at 2 h and then 10 h after the last meal. Discussion: This study therefore validates the optimization of metabolic programming by embryonic thermal manipulation for duck liver fattening. The understanding of the mechanisms of embryonic thermal programming in birds remains today very incomplete and the search for epigenetic marks (main hypothesis of the concept of programming) at the origin of the observed phenotypes could be the next step of this work.

Identification of different critical embryonic periods to modify egg incubation temperature in mule ducks.

Egg incubation of mule ducks, mainly used for fatty liver production, is one of the critical phases in this sector. Based on hatching rate, the best incubation parameters have already been well described for poultry, but the literature on ducks is lacking. In this study, we tested different incubation conditions by varying two important factors, temperature and relative humidity, in mule ducks. These variations were applied at different periods during embryogenesis in order to measure the impact of environmental disturbances on different zootechnical performances. The temperature was increased by 1.5 °C (16 h/24) and the relative humidity was set up to 65%, during 10 days. Six 10-day developmental windows were tested, from embryonic day 9 to embryonic day 14. Our results are in line with previous reports showing that increasing incubation temperature, even when relative humidity is adjusted, can have a negative impact on duck embryonic mortality up to 24.5% for the condition E10-E20 (P < 10-5). However, the hatchability can be maintained at the level of the control groups when these modifications are applied on the latest windows (from the 11th embryonic day). Sex ratio, hatching BW, and internal temperature are also sensitive to these incubation changes, and their modification could have a major impact on later zootechnical performance. These results should contribute to the development or embryonic temperature programming approaches, especially for the fatty liver production industry.

Application of TMAH thermochemolysis to the detection of nucleobases: Application to the MOMA and SAM space experiment.

Thermochemolysis of seven nucleobases-adenine, thymine, uracil, cytosine, guanine, xanthine, and hypoxanthine-in tetramethylammonium hydroxide (TMAH) was studied individually by pyrolysis gas chromatography mass spectrometry in the frame of the Mars surface exploration. The analyses were performed under conditions relevant to the Sample Analysis at Mars (SAM) instrument of the Mars Curiosity Rover and the Mars Organic Molecule Analyzer (MOMA) instrument of the ExoMars Rover. The thermochemolysis products of each nucleobase were identified and the reaction mechanisms studied. The thermochemolysis temperature was optimized and the limit of detection and quantification of each nucleobase were also investigated. Results indicate that 600°C is the optimal thermochemolysis temperature for all seven nucleobases. The methylated products trimethyl-adenine, 1, 3-dimethyl-thymine, 1, 3-dimethyl-uracil, trimethyl-cytosine, 1, 3, 7-trimethyl-xanthine (caffeine), and dimethyl-hypoxanthine, respectively, are the most stable forms of adenine, thymine, uracil, cytosine, guanine, and xanthine, and hypoxanthine in TMAH solutions. The limits of detection for adenine, thymine, and uracil were 0.075 nmol; the limits of detection for guanine, cytosine, and hypoxanthine were higher, at 0.40, 0.55, and 0.75 nmol, respectively. These experiments allowed to well constrain the analytical capabilities of the thermochemolysis experiments that will be performed on Mars to detect nucleobases.

Dietary methionine deficiency reduces laying performances of female common ducks and impacts traits of interest of their mule ducklings.

The effects of maternal nutrition on offspring phenotypes have been mainly documented over the past years in mammals, and are now studied in poultry as well. In the present study, we investigated the effects of a reduced level of dietary Methionine (Met) on laying performances of common laying ducks and their impacts on the phenotype of their mule ducklings. A total of 60 female laying ducks were divided into 2 dietary treatments at 10 wk of age. The restricted group received Met-restricted diets (R group) containing 0.25% of Met whereas the control group received control diets (C group) containing 0.40% of Met that meets Met requirements. The restriction was applied during the growing and laying periods, from 10 to 51 wk of age and a particular focus was put on female breeder traits that might be affected by the Met restriction. Plasma parameters of hepatic and lipid metabolisms were recorded in ducklings. Total weight (P < 0.001), albumen weight (P < 0.001) and albumen percentage of dry matter (P < 0.01) were decreased for eggs laid by female breeders from the R group. Both male and female ducklings from the R group of female breeders showed a reduced BW at hatching (P < 0.001) and a tendency to an increased proportional liver weight (P = 0.07). Finally, the maternal low dietary Met level modified plasma parameters in newborn ducklings regardless of sex: alkaline phosphatase (ALP) and alanine transaminase (ALT) activities were reduced (P = 0.07 and P = 0.002, respectively), levels of glucose (P = 0.03) and triglycerides (P = 0.01) were higher whereas level of free fatty acids decreased (P = 0.01). It was concluded that feeding female laying ducks with a restricted dietary Met content during the growing and laying periods has a negative effect on egg weight and composition. The ducklings that were restricted in nutrients during their early development, have a reduced BW, and altered lipid and hepatic metabolisms.

Contribution of radiation hybrids to genome mapping in domestic animals.

Radiation hybrid mapping has emerged in the end of the 1990 s as a successful and complementary approach to map genomes, essentially because of its ability to bridge the gaps between genetic and clone-based physical maps, but also using comparative mapping approaches, between 'gene-rich' and 'gene-poor' maps. Since its early development in human, radiation hybrid mapping played a pivotal role in the process of mapping animal genomes, especially mammalian ones. We review here all the different steps involved in radiation hybrid mapping from the constitution of panels to the construction of maps. A description of its contribution to whole genome maps with a special emphasis on domestic animals will also be presented. Finally, current applications of radiation hybrid mapping in the context of whole genome assemblies will be described.

The chicken RH map: current state of progress and microchromosome mapping.

The ChickRH6 radiation hybrid panel has been used to construct consensus chromosome radiation hybrid (RH) maps of the chicken genome. Markers genotyped were either from throughout the genome or targeted to specific chromosomes and a large proportion (one third) of data was the result of collaborative efforts. Altogether, 2,531 markers were genotyped, allowing the construction of RH reference maps for 20 chromosomes and linkage groups for four other chromosomes. Amongst the markers, 581 belong to the framework maps, while 1,721 are on the comprehensive maps. Around 800 markers still have to be assigned to linkage groups. Our attempt to assign the supercontigs from the chrun (virtual chromosome containing all the genome sequence that could not be attributed to a chromosome) as well as EST (Expressed Sequence Tag) contigs that do not have a BLAST hit in the genome assembly led to the construction of new maps for microchromosomes either absent or for which very little data is present in the genome assembly. RH data is presented through our ChickRH webserver (http://chickrh.toulouse.inra.fr/), which is a mapping tool as well as the official repository RH database for genotypes. It also displays the RH reference maps and comparison charts with the sequence thus highlighting the possible discrepancies. Future improvements of the RH maps include complete coverage of the sequence assigned to chromosomes, further mapping of the chrun and mapping of EST contigs absent from the assembly. This will help finish the mapping of the smallest gene-rich microchromosomes.

Assignment of non-informative turkey genetic markers through comparative approaches.

Molecular markers such as microsatellites, provide genetic signposts for navigating genomes. In general, genetic markers that are monomorphic or non-informative in mapping populations typically remain unmapped and as such are less likely to be included in future studies. The use of hybrid cell panels and in silico mapping via whole genome sequences allow for positional mapping of non-segregating markers. This study utilizes the INRA ChickRH6 whole-genome radiation hybrid panel and chicken whole-genome shotgun sequence to map microsatellite markers from the turkey (Meleagris gallopavo). Thirty-three of the 41 markers typed on the RH panel had significant linkage to at least one other marker and 83 of 100 sequences returned significant BLAST similarities. Positioning of these markers provides additional sequence tagged sites in the turkey genome and increases the potential use of these markers for future genetic studies.

A radiation hybrid map of chicken chromosome 15.

We have constructed a radiation hybrid (RH) map of chicken chromosome (GGA) 15. This map can be used as a resource to efficiently map genes to this chromosome. The map has been developed using a 6000 rad chicken-hamster whole-genome radiation hybrid panel (ChickRH6). In total, six microsatellite loci, 18 sequence tagged sites (STSs) from BAC end sequences and 11 genes were typed on the panel. The initial framework map comprised eight markers, and an additional 23 markers were then added to generate the final map. The total map length was 334 centiRay6000 (cR6000). The estimated retention frequency for the data set was 18%. Using an estimated physical length of 21 Mb, the ratio between cR6000 and physical distance over GGA15 was estimated to be 0.063 Mb/cR6000. The present map increases the marker density and the marker resolution on GGA15 and enables fast mapping of new chicken genes homologous to genes from human chromosomes 12 and 22.

Cloning of cDNA encoding the nuclear form of chicken sterol response element binding protein-2 (SREBP-2), chromosomal localization, and tissue expression of chicken SREBP-1 and -2 genes.

Sterol regulatory element binding protein-1 and -2 (SREBP-1 and -2) are key transcription factors involved in the biosynthesis of cholesterol and fatty adds. The SREBP have mainly been studied in rodents in which lipogenesis is regulated in both liver and adipose tissue. There is, however, a paucity of information on birds, in which lipogenesis occurs essentially in the liver as in humans. As a prelude to the investigation of the role of SREBP in lipid metabolism regulation in chicken, we sequenced the cDNA, encoding the mature nuclear form of chicken SREBP-2 protein, mapped SREBP-1 and -2 genes and studied their tissue expressions. The predicted chicken SREBP-2 amino acid sequence shows a 77 to 79% identity with human, mouse, and hamster homologues, with a nearly perfect conservation in all the important functional motifs, basic, helix-loop-helix, and leucine zipper (bHLH-Zip) region as well as cleavage sites. As in the human genome, SREBP-1 and SREBP-2 chicken genes are located on two separate chromosomes, respectively microchromosome 14 and macrochromosome 1. Tissue expression data show that SREBP-1 and SREBP-2 are expressed in a wide variety of tissues in chicken. However, unlike SREBP-2, SREBP-1 is expressed preferentially in the liver and uropygial gland, suggesting an important role of SREBP-1 in the regulation of lipogenesis in avian species.

Identification of three functions of the adenovirus e4orf6 protein that mediate p53 degradation by the E4orf6-E1B55K complex.

Complexes containing adenovirus E4orf6 and E1B55K proteins play critical roles in productive infection. Both proteins interact directly with the cellular tumor suppressor p53, and in combination they promote its rapid degradation. To examine the mechanism of this process, degradation of exogenously expressed p53 was analyzed in p53-null human cells infected with adenovirus vectors encoding E4orf6 and/or E1B55K. Coexpression of E4orf6 and E1B55K greatly reduced both the level and the half-life of wild-type p53. No effect was observed with the p53-related p73 proteins, which did not appear to interact with E4orf6 or E1B55K. Mutant forms of p53 were not degraded if they could not efficiently bind E1B55K, suggesting that direct interaction between p53 and E1B55K may be required. Degradation of p53 was independent of both MDM2 and p19ARF, regulators of p53 stability in mammalian cells, but required an extended region of E4orf6 from residues 44 to 274, which appeared to possess three separate biological functions. First, residues 39 to 107 were necessary to interact with E1B55K. Second, an overlapping region from about residues 44 to 218 corresponded to the ability of E4orf6 to form complexes with cellular proteins of 19 and 14 kDa. Third, the nuclear retention signal/amphipathic arginine-rich alpha-helical region from residues 239 to 253 was required. Interestingly, neither the E4orf6 nuclear localization signal nor the nuclear export signal was essential. These results suggested that if nuclear-cytoplasmic shuttling is involved in this process, it must involve another export signal. Degradation was significantly blocked by the 26S proteasome inhibitor MG132, but unlike the HPV E6 protein, E4orf6 and E1B55K were unable to induce p53 degradation in vitro in reticulocyte lysates. Thus, this study implies that the E4orf6-E1B55K complex may direct p53 for degradation by a novel mechanism.

Integration of chicken cytogenetic and genetic maps: 18 new polymorphic markers isolated from BAC and PAC clones.

As an approach to integrate the chicken genetic and cytogenetic maps, bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) clones were localized by fluorescence in situ hybridization (FISH) on chromosomes and by genetic mapping on the East Lansing and Compton reference families. Some of the clones used in this study were previously selected for the presence of potentially polymorphic (CA)n repeats and a microsatellite marker was developed when possible for genetic mapping. For other clones, a single strand conformational polymorphism (SSCP) was developed and used for this purpose. Between the two approaches, 18 markers linking the cytogenetic and genetic maps, seven on macrochromosomes and 11 on microchromosomes, were generated. Our results enabled the assignment and orientation of a linkage group to chromosome 3, together with the assignment of linkage groups to eight different microchromosomes, a fraction of the genome lacking mapping data and for which the degree of coverage by the genetic map was not well estimated previously.

Identification of 16 chicken microchromosomes by molecular markers using two-colour fluorescence in situ hybridization (FISH).

A feature of avian karyotypes is the presence of microchromosomes. As a typical avian genome, the chicken karyotype (2n = 78) consists of nine pairs of macrochromosomes, including the W and Z sexual chromosomes, and 30 pairs of indistinguishable microchromosomes usually ordered arbitrarily by decreasing size. Despite their reduced size, microchromosomes represent one-third of the genome and have a high gene density. So as to provide a tool to identify them, we developed a set of large insert-containing clones to be used as tags in two-colour fluorescence in situ hybridization experiments. Seventeen clones, six of which contain a microsatellite sequence and two others the fatty acid synthase gene or genes from the major histocompatibility complex, all presenting a strong hybridization signal, were selected for this purpose and enabled us to identify 16 different microchromosomes. The ability to recognize individual microchromosomes will be of great value for cytogenetic gene mapping, assignation of linkage groups from genetic maps and other studies on avian genome structure.

A deleted retroviral insertion at the ev21-K complex locus in Indonesian chickens.

Very poor feather development has been observed in chickens of the Nunukan strain, originating from Indonesia. The wing of the newly hatched chick does not show any primary or covert feathers; this phenotype will be referred to as very-late feathering (VLF). As adults, chickens are feathered but tail feathers are short and fragile. An experimental population was set up at the National Institute of Agronomic Research (INRA), Jouy-en-Josas, from one Nunukan male and four Nunukan females. Preliminary observations did not support the hypothesis of a sex-linked dominant mode of inheritance for the VLF phenotype. A restriction fragment length polymorphism (RFLP) study using five restriction enzymes and two probes, RAV-2 and endogenous virus (ev) ev21-int specific for the endogenous viral locus ALVE21, showed the presence of the expected 3' junction fragments for the ev21 occupied site but failed to reveal the expected 5' junction fragments for ev21 in Nunukan chickens. The unoccupied site corresponded to the ev21 unoccupied repeat (UR) of type a (URa). A deletion in the 5' region of the provirus and of the insertion site was indicated by the RFLP analysis and confirmed by a PCR study. Primers were designed in order to amplify a 5' junction fragment specific to the modified ev21 found in the Nunukan chickens. The sequence of this amplified product showed that the deletion started 652 bp upstream of the insertion site of ev21 and ended within the pol gene of the viral genome. This deletion represents a new allele, OSD, at the ev21 insertion site (locus ALVE21), that appears insufficient to produce a complete virus. Current data do not show a clear causal relationship between OSD and the VLF phenotype. The presence of OSD may be required but is not in itself sufficient to obtain the VLF phenotype. The genetic relationships between OSD and the altered feathering phenotype of Nunukan chickens will be investigated further in families segregating for the VLF phenotype, using the locus-specific PCR test developed as part of this study.

Associated effects of divergent selection for residual feed consumption on reproduction, sperm characteristics, and mitochondria of spermatozoa.

Eighteen generations of divergent selection for residual feed intake have been completed in two Rhode Island Red lines of domestic fowl. The high intake R+ line and the low intake R- line cocks used to sire Generation 19 of the selection experiment have been compared for associated responses on fertility, hatching, and sperm quality. Evaluations of sperm samples were based on volume, cell concentration, biochemical parameters (pH, uric acid and protein concentrations), and motility and morphology of spermatozoa. Finally, individual spermatozoa were analyzed by flow-cytometry (FCM) using Rhodamine 123 (Rh123) and nonyl-acrydine-orange (NAO) specific fluorochromes to assess, respectively, overall mitochondrial activity and overall mitochondrial content. Hatchability of incubated eggs was 20 points higher for the R- line, mainly because unfertilized eggs were only 6 vs 30% in the R+ line. Early embryo mortality was also twice as high in the R+ line (21%). The ratio of Rh123 to NAO fluorescence was identical for both lines. This result suggests that there was no difference in the energy producing potential of the individual mitochondria. Therefore, the difference seen for both dyes between the two lines might be attributed to a difference in the quantity of mitochondrial inner membranes present in the cell (with 17% less for the R+ line). In the R+ line, the poor performance at fertilization and during early embryonic development was associated with lower production of motile spermatozoa, possibly in relation to a lower quantity of mitochondria in spermatozoa from R+ cocks. Although the female contribution to the differences between lines was not explored separately, results suggest that selection for residual feed intake may have altered some cellular function related to the production of energy in the R+ line.