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.

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.

Performance comparison of laying hens segregating for the frizzle gene under thermoneutral and high ambient temperatures.

The effect on thermotolerance of the incompletely dominant frizzle (F) gene, which causes feather curling and feather mass reduction, was investigated in 281 laying hens that were homozygous for the frizzle mutation (FF), heterozygous (FN), or normally feathered (NN). One-half of the birds were kept under standard conditions (22°C) and half were exposed to high ambient temperatures (32°C) between 24 and 46 wk of age. Egg production, egg quality, feed efficiency, and dissection traits were recorded and compared. At standard conditions, egg production and quality traits did not differ among the 3 genotypes, whereas feed efficiency was lower for the homozygous birds. Under heat stress conditions, the superiority of the FF hens was evident for all egg quantity and quality traits. No significant difference was measured between heterozygous carriers and normally feathered hens, indicating that the incomplete dominant frizzle mutation behaved as a recessive mutation regarding heat tolerance. From this study, we deduced that the F mutation in its homozygous state has a beneficial effect in decreasing heat stress in poultry production, and it could be particularly advantageous in tropical countries where average temperatures are never too low to negatively affect feed efficiency.

Plumage colour mutations and melanins in the feathers of the Japanese quail: a first comparison.

The absorbance of melanin content from dorsal feathers was compared between wild-type Japanese quail and nine other quail plumage colours determined by single mutations in one of seven genes: extended brown (MC1R), yellow (ASIP), silver (MITF), lavender (MLPH), roux (TYRP1), imperfect albinism (SLC45A2) and rusty. As compared with wild-type quail, all mutations but extended brown decreased total melanins. The largest decrease was observed in quail with one of the dilution mutations at TYRP1, MLPH or SLCA45A2. No difference in eumelanins was found between the 10 plumage colours. Despite visible colour differences, homozygous and heterozygous mutants at MITF, or the two imperfect albino (white) and cinnamon (pale yellow) alleles at SLC45A2, could not be differentiated on the basis of melanins. In contrast, the two white phenotypes caused by mutations at MITF and SLC45A2, or the two reddish plumage colours caused by the roux and rusty non-allelic mutations had different total melanin contents. The results showed that rusty was not likely to be a dilution mutation.

Circulating corticosterone reaction to restraint and adrenocorticotropin hormone administration in white leghorns selected for immune response traits.

Corticosterone plasma concentration was measured in a random-bred control line and in 3 White Leghorn chicken lines previously selected over 9 generations for 3 different in vivo immune responses: high antibody response to Newcastle disease virus vaccine 3 wk after vaccination (ND3), high cell-mediated immune response (response to phytohemagglutinin, PHA), and high phagocytic activity measured as carbon clearance (CC). The objective of the study was to estimate if selection on immune response had an effect on the response to stress assessed by measures of corticosterone concentration before and after physical stress or adrenocorticotropin hormone injection and if the effect was dependent on the immune response trait that had been selected for, by joint analyses of immune responses and concentrations. The mean values of plasma corticosterone measures did not differ between lines, indicating that selection for different high immune responses had little effect on response to stress. Within line, however, significant negative correlations (-0.46

Delayed-type hypersensitivity response to KLH in F2 and backcrosses of two immune selected chicken lines: effect of immunisation and selection.

Cell-mediated immune response to keyhole limpet hemocyanin (KLH) was studied in 13 different progeny groups belonging to a second generation cross between two lines of White Leghorn previously selected for 10 generations for high antibody response to Newcastle disease vaccine (ND3) and high cell-mediated response to phytohemagglutinin (PHA) measured as wing web swelling. The cutaneous hypersensitivity response to KLH was assessed in KLH immunized and non-immunized birds. Wing thickness was measured at three time points: before the injection and 4 and 24 hours after the injection. The effect of previous immunization and genetic background was assessed. While no differences were present between progeny types or to a randomly bred control line at any of the three time points, significant differences were present between immunized and not immunized birds 24 hours after challenge. Results demonstrate that KLH immunized birds perform better and that the previous selection and crossbreeding scheme has not influenced the intensity of the skin swelling response to KLH. Apparently, neither selection for antibody responsiveness to ND3 nor selection for enhanced responsiveness to a T cell mitogen affected delayed-type hypersensitivity (DTH) to the Th 2 antigen KLH. These results may serve to facilitate further selection based on independent immune parameters.

Effect of introducing the naked neck gene in a line selected for low residual feed consumption on performance in temperate or subtropical environments.

The aim of this study was to investigate the possibility of combining the naked neck gene with a genetic background that has been optimized for feed efficiency of laying hens at moderate temperature. In 1997, the naked neck gene was introduced into a line selected for low residual feed intake. In 2003, after 6 generations of introgression, 8 males and 56 females heterozygous for the NA*NA mutation were used to generate all 3 genotypes (NA*N/NA*N, NA*NA/NA*N, NA*NA/NA*NA), in 2 hatches, for rearing in Taiwan and France, respectively, at 3-wk intervals. Growth performance, anatomical traits, laying traits, and feed efficiency were recorded in each country. In addition, comb and rectal temperatures were measured in Taiwan. Performance was generally lower and mortality of laying hens was higher in Taiwan (11%) than in France (1%). Genotype x environment interactions were rare: genotype x environment was observed only for body weight at 10 wk of age and was close to significance for egg weight. The laying performance was significantly decreased in Taiwan by about 25%. The naked neck genotype had a negative effect on body weight and a positive effect of clutch length and egg weight. It also affected heat dissipation traits such as wattle length and rectal and surface temperature measurements. There was a clear additive and negative effect of the NA*NA mutation on rectal temperature. Feed intake and residual feed intake were increased in the homozygous carriers of the NA*NA mutation, which may improve heat tolerance of the low residual feed consumption (R-) line. Feed efficiency tended to be better in NA*NA/NA*NA hens in both environments. Thus, the introduction of the NA*NA mutation in the R- background appeared to be favorable from the viewpoint of feed efficiency, but it did not improve laying performance in a subtropical environment. Other factors than temperature, such as diet composition and lighting regimen, may be involved.

Association of a single-nucleotide substitution in TYRP1 with roux in Japanese quail (Coturnix japonica).

We investigated TYRP1 as a candidate locus for the recessive, sex-linked roux (br(r)) phenotype in Japanese quail. A screen of the entire coding sequence of TYRP1 in roux and wild-type quail revealed a non-synonymous T-to-C substitution in exon 3, leading to a Phe282Ser mutation. This was perfectly associated with plumage phenotype: all roux birds were homozygous for Ser282. Co-segregation of the Phe282Ser mutation with the roux phenotype was confirmed in three br(r)/BR+ x br(r)/- backcrosses. We found no significant difference in TYRP1 expression between roux and wild-type birds, suggesting that this association is not due to linkage disequilibrium with an unknown regulatory mutation. In addition, the Phe282 amino acid appears to be of functional significance, as it is highly conserved across the vertebrates. This is the first demonstration that TYRP1 has a role in pigmentation in birds.

Effects of the dominant lethal yellow mutation on reproduction, growth, feed consumption, body temperature, and body composition of the Japanese quail.

Wild-type Japanese quail were compared with full-sibs with a yellow plumage color determined by an autosomal dominant mutation (Y), which is lethal when homozygous. These quail have wheat-straw yellow-colored feathers. Early growth was slower in yellow quail that had 2.4% lower BW than wild-type quail (149.3 g vs. 153.0 g) at 28 d of age. The BW, however, was similar for yellow and wild-type males at 35 d, and it remained so throughout the last part of the growth of the quail monitored until the age of 120 d, as indicated by the very close parameters of the monomolecular growth curve [BW= A - B exp(-kt)] obtained for the 2 groups. Yellow plumage color was also associated with a more difficult adaptation to housing (measured by temporary BW loss) in individual cages and to a significantly 0.2 degrees C lower body temperature at 42 d, but feed consumption and residual feed intake were similar for the 2 plumage color phenotypes. Breast and liver weights were similar in the 2 groups, but abdominal fat was 24% higher (4.66 vs. 3.76 g) in yellow quail. There is some association between the correlated effects of the Y gene in quail and those of the lethal mutation A(y) at the agouti locus in the mouse.

Antibody responses to keyhole limpet hemocyanin, lipopolysaccharide, and Newcastle Disease virus vaccine in F2 and backcrosses of white Leghorn lines selected for two different immune response traits.

Planned crosses were designed to produce an F(2) and 2 backcross populations from 2 lines of White Leghorn chickens previously selected over 10 generations for 2 different in vivo immune responses. The selection criteria applied on the 2 grandparental lines were as follows: high antibody response to Newcastle disease virus vaccine 3 wk after vaccination (ND3) and high cell-mediated immune response [response to phytohemagglutinin]. Furthermore a control line was kept by random breeding. The objective of the study was to estimate if the 2 selection criteria applied on the pure lines had changed the level of and type of immune (humoral) response to a new antigen, keyhole limpet hemocyanin (KLH), in the various second-generation progeny groups. In addition, correlations between parameters of acquired and innate immunity were tested. Primary total (IgT) and isotype-specific (IgG and IgM) antibody response to KLH 1 wk after immunization and levels of natural antibodies (NAB) binding to Salmonella enteriditis-derived lipopolysaccharide (LPS) were measured. Although no differences were present between IgM and IgG antibodies to KLH and the phytohemagglutinin skin-swelling response, significant differences were present between all the progeny groups for IgT to KLH and ND3 and NAB binding to LPS. The mean values for IgT to ND3 and KLH were significantly different between the crosses using the selected lines compared with the control line, indicating a contribution of the previous selection. In addition, a sex effect was found for IgM to KLH and NAB to LPS, for which females had a higher response than males in both cases. No interaction between progeny type and sex was found. Furthermore, significant positive correlations were found between NAB to LPS and specific antibody titers to KLH. Finally, the results of the present study demonstrated an interaction between innate and acquired immunity under this strategy of selection and crossbreeding and confirmed the effect of selection on general immune response to a new antigen in second-generation crosses.

Quantitative effects of an intronic retroviral insertion on the transcription of the tyrosinase gene in recessive white chickens.

Recently, we reported the complete association of a retroviral insertion in intron 4 of the tyrosinase gene and the recessive white mutation (c) in chickens. The mutant allele carrying the retroviral insertion produced, in skin samples of 10-week-old chickens, aberrant tyrosinase transcripts that did not contain exon 5. In the present study, we performed serial molecular and statistical analyses on embryos and 10-week-old chickens to characterize the quantitative effect of the retroviral insertion on the expression pattern of tyrosinase in different tissues (skin and retina). By using quantitative real-time RT-PCR, we observed that the expression level of tyrosinase was significantly lower in recessive white chickens than in wild-type coloured chickens, but that this pattern was age- and tissue-dependent. The differential expression in skin was not significant in embryos, whereas it was highly significant in 10-week-old chickens. Furthermore, there was no difference in the expression of tyrosinase in the retinal pigment epithelium of animals with different genotypes; this corresponds to phenotypic data, which show pigmented eyes in both genotypes. These findings show that the retroviral insertion disturbs tyrosinase expression in the recessive white mutant chickens, and suggests that the regulation of tyrosinase expression in chickens differs between embryos and growing animals, as well as between skin and retina.

Semen cryopreservation for ex situ management of genetic diversity in chicken: creation of the French avian cryobank.

The need for semen preservation in domestic birds is a result of the reduction in genetic variability of domestic bird livestock and of the increasing risk of line extinction for health and safety reasons. Cryopreservation of embryos and primordial germ cells (PGC) is not routinely feasible in birds. The project therefore involved semen frozen in optimal safety and traceable conditions. Whole blood samples were also frozen to provide samples of analyses of genomes and health status. The feasibility of using ex situ conservation, i.e., collecting biological material to be stored outside the usual production area of the species (ex situ genetic stock), to preserve and manage rare breeds was tested with 4 subfertile populations: 3 rare experimental lines used for research into energy metabolism (R+), growth (Y33), and immunity (B4/B4), reared under known health status and the oldest endangered patrimonial French breed, the Gauloise dorée with an unknown health status. A general infrastructure was set up for the health screening and remediation of diseases, collection and storage of frozen cells and 2 sites were created for the storage of frozen samples. The screening and remediation of diseases of the Gauloise dorée, which was contaminated with various Salmonella and Mycoplasma strains, was achieved by successive treatment of parents, incubated eggs and young chicks with Baytril followed by Tiamulin. For each line, 474 to 994 semen straws have been frozen, thawed, and the semen evaluated. Insemination of frozen-thawed semen into females of the same genetic origin or of an egg-type commercial breed produced chicks in every case. For the most subfertile lines, insemination with egg-type females significantly increased the reproductive success. In conclusion, we report on the benefits of a semen and blood cryobanking complex for the management of endangered lines and strains of domestic birds. Current stocks made possible the restoration of more than 96% of the initial genome. This project also provided technical solutions to resolve some of the health problems frequently encountered for gene preservation in poultry.

Development and performances of a Japanese quail line homozygous for the diabetes insipidus (di) mutation.

A strain of Japanese quail with the polyuria disorder (excessive urination) was developed from founders that regurgitated water spontaneously. A back-cross with a nonpolyuric quail line showed that the polyuric strain was fixed for an autosomal recessive mutation that also induced polydipsia (excessive drinking). Plasma levels and brain mRNA contents for avian Arg vasotocin were little affected by the mutation, but plasma avian Arg vasotocin was 13-fold higher and brain mRNA contents were significantly increased in both normal and mutant quail following a 24-h water deprivation. Affected and normal birds had similar performance traits (egg production and quality, feed intake, and gross carcass traits), but residual feed consumption was higher in polydipsic males. These results are consistent with the hypothesis that this strain was fixed for a mutation similar to the di gene described in the chicken and which induces nephrogenic diabetes insipidus. This new strain of Japanese quail might constitute a convenient model for the analysis of the underlying mechanisms of the disorder in birds and for comparative study with mammals.

Effect of high ambient temperature and naked neck genotype on performance of dwarf brown-egg layers selected for improved clutch length.

1. Two experimental lines of dwarf brown-egg layers selected on clutch length were reciprocally crossed. In total, 288 hens, from three genotypes: L1, purebred normally feathered (121 hens), F1, crossbred and heterozygous for the NA mutation (99 hens) or L2, purebred homozygous for NA (68 hens) were housed in three climatic rooms at 22 degrees C and three climatic rooms at 32 degrees C. 2. Body weight, daily egg production records, feed intake and some anatomical and physiological traits were recorded. Canonical discriminant analysis was used to examine the relationships among all measured traits and to compare the experimental groups as defined by genotype and temperature. 3. Heterosis effects were observed on body weight, feed intake, egg mass and some anatomical traits at 22 degrees C, but were negligible for most of the traits measured at 32 degrees C except for body weight at 34 weeks, clutch length and some anatomical traits. 4. Genotype by temperature interactions were observed for egg production, egg mass and feed intake. The temperature stress was severe, with a marked decrease in egg production. The naked neck gene could limit the negative effect of long-term heat stress. Egg weight was increased by the NA gene, as usually observed in layers. 5. The first three canonical variates explained altogether 97.4% of the variance. The first canonical variate was associated almost exclusively with ambient temperature. The second was associated with genotype. The third was associated with heterozygous genotype for NA mutation and genotype by temperature interaction. Stepwise discriminant analysis indicated that 12 traits out of 20 were effective at detecting the differences among the genotype and temperature combinations.

Effects of two-locus combinations, using the roux, lavender, and beige mutations, on plumage color of Japanese quail.

The interactions between the effects of three plumage color mutations taken two-by-two (sex-linked recessive roux, autosomal recessive lavender, and autosomal dominant beige) were studied in Japanese quail by producing a total of 121 F(1) and 1118 F(2) quail from the three pure stocks. Three new plumage colors were obtained in F(2) quail: roux-diluted beige, cream, and lavender-diluted beige. Two of them, roux-diluted beige (from the roux and beige mutations) and cream (from the roux and lavender mutations) corresponded to double homozygotes or hemizygous birds, and could therefore be used to tag a quail line. On the other hand, an F(3) from F(2) birds with lavender-diluted beige plumage was necessary to show that quail with this plumage color were homozygous for the lavender mutation, but were either homozygous or heterozygous for the beige gene. In all three F(2)s, observed segregation of plumage colors fit simple two-locus Mendelian inheritance.

Testing homology of loci for two plumage colors, "lavender" and "recessive white," with chicken and Japanese quail hybrids.

Homology for two plumage color loci was studied by hybridization between chickens and Japanese quail. First, chicken-quail hybrids were produced from homozygous "lavender" chicken cocks and "bleu" Japanese quail, and all 30 hybrids had the same parental slate blue plumage color. On the other hand, no hybrids with this plumage were obtained out of 18 progeny from the same cocks and wild-type quail. These results show that the slate blue plumage color is determined by homologous loci in Japanese quail and chickens. Second, all (n = 25) chicken-quail hybrids hatched from homozygous "recessive white" cocks and "recessive white" (n = 8) or "wild-type" (n = 17) quail had the same pattern of plumage color, with white feathers on the ventral face and colored feathers elsewhere. These results indicate that the recessive white mutations are not homologous in Japanese quail and chickens.

Fearfulness and performance related traits in selected lines of Japanese quail (Coturnix japonica).

Fearfulness and economic traits were studied in three lines of Japanese quail. Two of the lines were of the same genetic origin and were subjected to divergent selection for the duration of tonic immobility (TI), a measure of fearfulness. Birds were selected for long (LTI) or short (STI) duration of TI. The third line (DD) was of a different genetic origin and had been selected for early egg production. Fear, growth, residual feed intake, and measures of egg composition and production varied among lines. The distribution of TI in Line DD was closer to that from Line STI. Residual feed intake and shell content were lowest in the DD line. The DD birds laid more broken eggs than quail of the other lines. The STI line birds had higher BW and laid more, but smaller, eggs than LTI line birds. Eggs laid by LTI line birds had higher albumen content, but lower percentage shell, than those laid by STI line birds. When all traits were considered together, there was an overall tendency for STI line birds to out perform LTI birds with DD line birds showing intermediate performance. This finding supports the notion that there is a relationship between fearfulness and productivity. However, the skewed distribution of TI precluded estimation of correlation with production traits in the LTI and STI lines. No significant relationships among fear and production-related traits were found in the DD line, which contradicts the notion that fearfulness and production are related.