Chicken adaptive response to low energy diet: main role of the hypothalamic lipid metabolism revealed by a phenotypic and multi-tissue transcriptomic approach.

BACKGROUND: Production conditions of layer chicken can vary in terms of temperature or diet energy content compared to the controlled environment where pure-bred selection is undertaken. The aim of this study was to better understand the long-term effects of a 15%-energy depleted diet on egg-production, energy homeostasis and metabolism via a multi-tissue transcriptomic analysis. Study was designed to compare effects of the nutritional intervention in two layer chicken lines divergently selected for residual feed intake. RESULTS: Chicken adapted to the diet in terms of production by significantly increasing their feed intake and decreasing their body weight and body fat composition, while their egg production was unchanged. No significant interaction was observed between diet and line for the production traits. The low energy diet had no effect on adipose tissue and liver transcriptomes. By contrast, the nutritional challenge affected the blood transcriptome and, more severely, the hypothalamus transcriptome which displayed 2700 differentially expressed genes. In this tissue, the low-energy diet lead to an over-expression of genes related to endocannabinoid signaling (CN1R, NAPE-PLD) and to the complement system, a part of the immune system, both known to regulate feed intake. Both mechanisms are associated to genes related polyunsaturated fatty acids synthesis (FADS1, ELOVL5 and FADS2), like the arachidonic acid, a precursor of anandamide, a key endocannabinoid, and of prostaglandins, that mediate the regulatory effects of the complement system. A possible regulatory role of NR1H3 (alias LXRα) has been associated to these transcriptional changes. The low-energy diet further affected brain plasticity-related genes involved in the cholesterol synthesis and in the synaptic activity, revealing a link between nutrition and brain plasticity. It upregulated genes related to protein synthesis, mitochondrial oxidative phosphorylation and fatty acid oxidation in the hypothalamus, suggesting reorganization in nutrient utilization and biological synthesis in this brain area. CONCLUSIONS: We observed a complex transcriptome modulation in the hypothalamus of chicken in response to low-energy diet suggesting numerous changes in synaptic plasticity, endocannabinoid regulation, neurotransmission, lipid metabolism, mitochondrial activity and protein synthesis. This global transcriptomic reprogramming could explain the adaptive behavioral response (i.e. increase of feed intake) of the animals to the low-energy content of the diet.

Genetic structure of Eurasian and North American mallard ducks based on mtDNA data.

To elucidate the origin and genetic structure of the domesticated duck in Eurasia and North America, we sequenced 114 duck D-loop sequences and retrieved 489 D-loop sequences from GenBank. In total, 603 ducks including 50 duck breeds/populations from eight countries (China, France, Russia, India, Kazakhstan, Mongolia, Thailand and USA) were used in this study. One hundred and thirty-four haplotypes and 81 variable sites were detected. H49 was the predominant haplotype, which was considered to be the same dominant haplotype found in the previous studies, and was found in 309 birds. The smallest values for both genetic differentiation index (F(ST), 0.04156) and the number of the net nucleotide substitutions between two populations (D(A), 0.00018) were observed between Eurasian domestic ducks and Eurasian mallards. No geography, breed or population clusters were observed in the Eurasian domestic ducks and mallards. Five haplotypes were shared by USA mallards and Eurasian domestic duck/Eurasian mallards. Only one haplotype (H49) was shared by Eurasian domestic ducks and China spot-billed ducks. By combining phylogenetic analyses, haplotype network profile, genetic distances and shared haplotypes, we can draw two major conclusions: (i) Eurasian and North American mallards show a clear geographic distribution pattern; (ii) Eurasian domestic ducks are derived from the Eurasian mallards, not from the spot-billed ducks.

Quantitative trait loci for resistance to infection in sheep using a live Salmonella Abortusovis vaccine.

Quantitative trait loci (QTL) mapping for susceptibility to a Salmonella Abortusovis vaccinal strain was performed using an experimental design involving 30 Romane sheep sire families (1216 progenies). Nine QTL corresponding to bacterial load, weight variations and antibody response criteria were mapped on eight chromosomes, including the major histocompatibility complex area on chromosome 20. Surprisingly, none was found to be significant in the SLC11A1 region (formerly NRAMP1) that has been shown to influence Salmonella susceptibility in other species.

Short communication: Effects of in-ovo injection of endocrine disruptors and methyltransferase inhibitor on quail growth and egg-laying performances.

Early experiences, including prenatal environment, are known to influence a wide variety of mechanisms involved in the phenotype elaboration. We investigated the effect of the addition of endocrine disruptors or of a methyltransferase inhibitor during the embryonic development of quails from different genetic backgrounds (four different quail lines) on their growth and egg-laying performances. Fifty-four pairs of parents per line were used and fertilised eggs from each pair were randomly divided into five groups: a control group without any injection, an injected control group treated by injection into the egg of sesame oil, and three groups treated by injection of Genistein, Bisphenol A or 5-Aza-2'-deoxycytidine. All quails were individually weighed at 8, 21, 36 and 78 days. The age at first egg laid and the number of eggs laid were recorded. These analyses revealed a significant impact of the treatment on growth but no influence on the egg-laying traits. All three molecules significantly affected at least one of the analysed growth traits. In conclusion, we showed that the injection of endocrine disruptors or DNA methyltransferase inhibitor into the egg had significant effects on quail development; these effects were specific to each treatment, but no interaction between line and treatment was observed.

Mapping QTL for growth and shank traits in chickens divergently selected for high or low body weight.

An F(2) population (695 individuals) was established from broiler chickens divergently selected for either high (HG) or low (LG) growth, and used to localize QTL for developmental changes in body weight (BW), shank length (SL9) and shank diameter (SD9) at 9 weeks. QTL mapping revealed three genome-wide QTL on chromosomes (GGA) 2, 4 and 26 and three suggestive QTL on GGA 1, 3 and 5. Most of the BW QTL individually explained 2-5% of the phenotypic variance. The BW QTL on GGA2 explained about 7% of BW from 3 to 7 weeks of age, while that on GGA4 explained 15% of BW from 5 to 9 weeks. The BW QTL on GGA2 and GGA4 could be associated with early and late growth respectively. The GGA4 QTL also had the largest effect on SL9 and SD9 and explained 7% and 10% of their phenotypic variances respectively. However, when SL9 and SD9 were corrected with BW9, a shank length percent QTL was identified on GGA2. We identified novel QTL and also confirmed previously identified loci in other chicken populations. As the foundation population was established from commercial broiler strains, it is possible that QTL identified in this study could still be segregating in commercial strains.

QTL for resistance to Salmonella carrier state confirmed in both experimental and commercial chicken lines.

The ability of chickens to carry Salmonella without displaying disease symptoms is responsible for Salmonella propagation in poultry stocks and for subsequent human contamination through the consumption of contaminated eggs or meat. The selection of animals more resistant to carrier state might be a way to decrease the propagation of Salmonella in poultry stocks and its transmission to humans. Five QTL controlling variation for resistance to carrier state in a chicken F(2) progeny derived from the White Leghorn inbred lines N and 6(1) had been previously identified using a selective genotyping approach. Here, a second analysis on the whole progeny was performed, which led to the confirmation of two QTL on chromosomes 2 and 16. To assess the utility of these genomic regions for selection in commercial lines, we tested them together with other QTL identified in an [Nx6(1)] x N backcross progeny and with the candidate genes SLC11A1 and TLR4. We used a commercial line divergently selected for either low or high carrier-state resistance both in young chicks and in adult hens. In divergent chick lines, one QTL on chromosome 1 and one in the SLC11A1 region were significantly associated with carrier-state resistance variations; in divergent adult lines, one QTL located in the major histocompatibility complex on chromosome 16 and one in the SLC11A1 region were involved in these variations. Genetic studies conducted on experimental lines can therefore be of potential interest for marker-assisted selection in commercial lines.

A comprehensive analysis of QTL for abdominal fat and breast muscle weights on chicken chromosome 5 using a multivariate approach.

Quantitative trait loci (QTL) influencing the weight of abdominal fat (AF) and of breast muscle (BM) were detected on chicken chromosome 5 (GGA5) using two successive F(2) crosses between two divergently selected 'Fat' and 'Lean' INRA broiler lines. Based on these results, the aim of the present study was to identify the number, location and effects of these putative QTL by performing multitrait and multi-QTL analyses of the whole available data set. Data concerned 1186 F(2) offspring produced by 10 F(1) sires and 85 F(1) dams. AF and BM traits were measured on F(2) animals at slaughter, at 8 (first cross) or 9 (second cross) weeks of age. The F(0), F(1) and F(2) birds were genotyped for 11 microsatellite markers evenly spaced along GGA5. Before QTL detection, phenotypes were adjusted for the fixed effects of sex, F(2) design, hatching group within the design, and for body weight as a covariable. Univariate analyses confirmed the QTL segregation for AF and BM on GGA5 in male offspring, but not in female offspring. Analyses of male offspring data using multitrait and linked-QTL models led us to conclude the presence of two QTL on the distal part of GGA5, each controlling one trait. Linked QTL models were applied after correction of phenotypic values for the effects of these distal QTL. Several QTL for AF and BM were then discovered in the central region of GGA5, splitting one large QTL region for AF into several distinct QTL. Neither the 'Fat' nor the 'Lean' line appeared to be fixed for any QTL genotype. These results have important implications for prospective fine mapping studies and for the identification of underlying genes and causal mutations.

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.

An integrated approach of genetic resistance to Salmonella carrier state in fowls: from genetics to genomics and modelling.

Increasing resistance to acute Salmonellosis (that is, contamination level shortly after infection) is not sufficient to reduce the risk for consumers to be contaminated by Salmonella. Indeed, animals may remain contaminated at a low level for weeks or months. Increased resistance to the Salmonella carrier state, i.e., animals' ability to clear bacteria, is needed; it involves measuring bacterial contamination several weeks after inoculation with a low dose. To study such resistance traits, three convergent approaches were used. A quantitative trait loci (QTL) study was performed, taking advantage of inbred lines differing in resistance. Several QTLs controlling resistance at a younger age were identified and are currently being confirmed in a new cross before finer mapping, using advanced intercross lines. These inbred lines are also presently being compared using functional genomics. In parallel, a selection experiment for increased or decreased resistance at a younger and a later age was undertaken. Besides providing genetic models differing in their levels of resistance, it underlined the importance of the choice of selection criterion, whether marker assisted or not. Indeed, genes controlling resistance are strongly dependant on age; selecting for resistance at a younger age might result in increased susceptibility at an older age. Finally, the results of this experiment were used in a model of the intra-flock propagation of Salmonella. It showed that introducing a proportion of resistant animals within a flock of susceptible hens could dramatically change the evolution of contamination. Moreover, it demonstrated the magnitude of synergy between selection and vaccination, which should enhance the interest of increased resistance. The results show that selection for increased resistance to the Salmonella carrier state may be efficient, providing that the appropriate criteria of selection are used.

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.

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.

Effect of two candidate genes on the Salmonella carrier state in fowl.

Selection for increased resistance to Salmonella carrier-state (defined as the persistency of the bacteria 4 wk after inoculation) could reduce the risk for the consumer of food toxi-infections. The effects of two genomic regions on chromosomes 7 and 17 harboring two genes, NRAMP1 (SLC11A1) and TLR4, known to be involved in the level of chicken infection 3 d after inoculation by Salmonella were thus tested on a total of 331 hens orally inoculated at the peak of lay with 10(9) bacteria. The animals and their parents were genotyped for a total of 10 microsatellite markers mapped on chromosomes 7 and 17. Using maximum likelihood analysis and interval mapping, it was found that the SLC11A1 region was significantly involved in the control of the probability of spleen contamination 4 wk after inoculation. Single nucleotide polymorphisms (SNP) within the SLC11A1 and TLR4 gene were tested on those animals as well as on a second batch of 279 hens whose resistance was assessed in the same conditions. As the former was significantly associated with the risk of spleen contamination and the number of contaminated organs, SLC11A1 appears to be involved in the control of resistance to Salmonella carrier state. The involvement of the TLR4 gene was also highly suspected as a significant association between SNP within the gene, and the number of contaminated organs was detected.

A genome scan with AFLP markers to detect fearfulness-related QTLs in Japanese quail.

A quantitative trait loci (QTL) study was undertaken to identify genome regions involved in the control of fearfulness in Japanese quail (Coturnix japonica). An F2 cross was made between two quail lines divergently selected over 29 generations on duration of tonic immobility (DTI), a catatonic-like state of reduced responsiveness to a stressful stimulation. A total of 1065 animals were measured for the logarithm of DTI (LOGTI), the number of inductions (NI) necessary to induce the immobility reaction, open-field behaviour including locomotor activity (MOVE), latency before first movement (LAT), number of jumps (JUMP), dejections (DEJ) and shouts (SHOUT), corticosterone level after a contention stress (LOGCORT) and body weight at 2 weeks of age (BW2). A total of 310 animals were included in a genome scan using selective genotyping with 248 AFLP markers. A total of 21 suggestive or genome-wide significant QTL were observed. Two highly significant QTL were identified on linkage group 1 (GL1), one for LOGTI and one for NI. In the vicinity of the QTL for LOGTI, a nearly significant QTL for SHOUT and a suggestive QTL for LAT were also identified. On GL3, genome-wide significant QTL were observed for JUMP and DEJ as well as suggestive QTL for LOGTI, MOVE, SHOUT and LAT. A significant QTL for BW2 was observed on GL2 and a nearly significant one on GL1. These results may be useful in the understanding of fearfulness in quail and related species provided that fearfulness has the same genetic basis.

A linkage map with microsatellites isolated from swine flow-sorted chromosome 11.

We have developed a simple and efficient method to construct partial libraries of swine Chromosome (Chr) 11, starting with only 300 flow-sorted copies. DNA is amplified by PARM-PCR with primer containing at the 5'-end the sequence AGCU-. After amplification, digestion of PCR products with uracil DNA glycosylase generates cohesive ends corresponding to the SstI site. The amplified fragments can then be ligated in vector linearized with the SstI enzyme. Using five different primers, we PARM-PCR amplified and cloned swine Chr 11 DNA. These chromosome-specific libraries have been used to develop 14 different (TG)n microsatellites. Ten of these markers were assigned to Chr 11 by PCR analysis of a panel of Pig-Rodent somatic hybrids and by linkage analysis of the 171 individuals of the PiGMaP reference families. A complete linkage map of 147 cM of this chromosome was then realized by integrating existing markers.

Mapping the naked neck (NA) and polydactyly (PO) mutants of the chicken with microsatellite molecular markers.

The bulked segregant analysis methodology has been used to map, with microsatellite markers, two morphological mutations in the chicken: polydactyly (PO) and naked neck (NA). These autosomal mutations show partial dominance for NA, and dominance with incomplete penetrance for PO. They were mapped previously to different linkage groups of the classical map, PO to the linkage group IV and NA being linked to the erythrocyte antigen CPPP. An informative family of 70 offspring was produced by mating a sire, heterozygous for each of the mutations, to 7 dams homozygous recessive for each locus. Three DNA pools were prepared, pool PO included 20 chicks exhibiting at least one extra-toe, pool NA included 20 non-polydactyly chicks showing the typical phenotype associated with heterozygosity for the naked neck mutation, and pool NP included 20 chicks exhibiting neither of the mutant phenotypes. Typings were done on an ABI-373 automatic sequencer with 147 microsatellite markers covering most of the genome. An unbalanced distribution of sire marker alleles were detected between pool PO, and pools NA and NP, for two markers of chromosome 2p, MCW0082 and MCW0247. A linkage analysis taking into account the incomplete penetrance of polydactyly (80% ) was performed with additional markers of this region and showed that the closest marker to the PO locus was MCW0071 (5 cM, lod score = 9). MCW0071 lies within the engrailed gene EN2 in the chicken. In the mouse, the homologous gene maps on chromosome 5, close to the hemimelic extra-toes mutation Hx. In the case of the NA locus, markers of chromosome 3 were selected because CPPP was mapped on this chromosome. Analysis of individual typings showed a linkage of 5.7 cM (lod score = 13) between the NA locus and ADL0237 in the distal region of chromosome 3q. These results contribute to connecting the former classical map to the molecular genetic map of the chicken, and open the way to the identification of the molecular nature of two developmental mutations of the chicken that are known to occur in many breeds of chickens.

Regional localization of the ovine NRAMP gene to chromosome 2q41-->q42 by in situ hybridization.

The murine Ity/Lsh/Bcg locus belongs to a conserved region between mouse Chromosome 1 and human chromosome 2. This gene governs mouse resistance to several intracellular pathogens. We have localized the gene on sheep chromosome 2q41-->q42 by radioactive in situ hybridization using a 2.1-kb fragment of the ovine equivalent of this gene.

cDNA cloning, structural organization, and expression of the sheep NRAMP1 gene.

Mouse resistance to several intracellular pathogens including Mycobacteria, Leishmania, and Salmonella is under the control of the Chromosome (Chr) 1 Natural Resistance Associated Macrophage Protein I gene (Nramp1). This gene could have an economic and health importance for domestic animals and humans as well. Therefore, equivalents of the NRAMP1 gene have been cloned by several research groups in various animal species. To study in sheep the influence of the NRAMP1 gene on the susceptibility to intracellular pathogens induced diseases, we have cloned the sheep NRAMP1 cDNA by screening a splenic cDNA library. The genomic organization of the sheep NRAMP1 gene was then determined by sequencing the exon/intron boundaries. The transcription start points (tsp) from the NRAMP1 mRNA have been located with primer extension experiments. RT-PCR reactions have been used to determine the profile of mRNA expression of this gene.

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.

Mapping of FASN and ACACA on two chicken microchromosomes disrupts the human 17q syntenic group well conserved in mammals.

Fatty acid synthase and Acetyl-CoA carboxylase are both key enzymes of lipogenesis and may play a crucial role in the weight variability of abdominal adipose tissue in the growing chicken. They are encoded by the FASN and ACACA genes, located on human Chromosome (Chr) 17q25 and on Chr 17q12 or 17q21 respectively, a large region of conserved synteny among mammals. We have localized the homologous chicken genes FASN and ACACA coding for these enzymes, by single-strand conformation polymorphism analysis on different linkage groups of the Compton and East Lansing consensus genetic maps and by FISH on two different chicken microchromosomes. Although synteny is not conserved between these two genes, our results revealed linkage in chicken between FASN and NDPK (nucleoside diphosphate kinase), a homolog to the human NME1 and NME2 genes (non-metastatic cell proteins 1 and 2), both located on human Chr 17q21.3, and also between FASN and H3F3B (H3 histone family 3B), located on human Chr 17q25. The analysis of mapping data from the literature for other chicken and mammalian genes indicates rearrangements have occurred in this region in the mammalian lineage since the mammalian and avian radiation.