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.

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.

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.

Heterosis in normal versus dwarf laying hens.

The effect of genotype at the sex-linked dwarf locus on heterosis in crosses between a White Leghorn and a brown egg line for body weight, egg production, and related traits was studied. Heterozygous Dw/dw males were used to produce normal and dwarf pullets in each of the pure lines and their reciprocal crosses (eight genotype-line combinations). There were 54 pullets per combination. Line differences were significant for shank length, body weights at 8, 17, and 52 wk, age at first egg, egg number, clutch length, rate of lay, and egg weight. Heterosis was observed for all of these traits. Body weight as a covariate was not important in analyses of egg number, clutch length, and egg weight. The egg production reduction associated with the dw gene in pure lines was smaller in F1 hens. This discovery may be adequate to warrant use of dwarf crossbred hens for egg production.

A naturally occurring growth hormone receptor mutation: in vivo and in vitro evidence for the functional importance of the WS motif common to all members of the cytokine receptor superfamily.

To obtain an animal model for studying the role of the GH receptor (GHR) in growth and development, we analyzed a sex-linked dwarf (SLD) chicken strain (Leghorn) which exhibits phenotype similarities with a human genetic growth disorder, an autosomal recessive GH resistance condition (Laron dwarfism). Having previously demonstrated the responsibility of the human GHR gene in the Laron phenotype, we focused our analysis on the corresponding gene in SLD chickens. Sequencing of the whole coding region of the chicken GHR cDNA identified a G-to-T transversion segregating with the SLD phenotype and generating an isoleucine instead of a serine at position 199 within a highly conserved region close to the junction between the extracellular and transmembrane domains. This defect involves the last invariant amino acid of the WS-like motif (amino acid sequence WSXWS) common to all members of the cytokine receptor superfamily. Transfection of a mutated GHR cDNA containing this mutation into eukaryotic cells led to the synthesis of a receptor protein that displayed impaired plasma membrane expression and binding activity. These data define the molecular basis for the SLD phenotype and identify this strain as an interesting model for studying Laron dwarfism in humans; this animal model may also represent a system in which therapeutic strategies to promote growth can be evaluated. Finally, the nature of the molecular defect identified provides direct evidence for the functional importance of the WS motif in GHRs and related receptors.

Differential environmental effects on lesions, early growth, and mortality of imperfect albino (Sal-c) chicks.

A series of experiments investigated early pleiotropic effects of a gene for imperfect albinism (s(al-c) in a population of chickens at Jouy-en-Josas, France. An elevated incidence of lesions of the navel, hocks, and nares typical of imperfect albinos were seen on these chicks, confirming their existence in this population. Variations in hatching environment and the amount of light in the hatcher both implicated in contributing to the occurrence of lesions of the hocks and nares, but not to those of the naval. In two experiments, using batteries and cages, early growth was reduced among albinos and early mortality was increased. Significant genotype by environment interactions for weight at 4 days and growth to this time, but not thereafter, suggested that the effect is restricted to this time. Unfavorable environments were most deleterious to albinos. In a third experiment, conducted in floor pens, the gene had no effect on either early growth or mortality. There was no effect of the gene on the lesions, early growth, or mortality when carried by heterozygous males (s+/s(al-c)).

Effects of insulin-like growth factor-I (IGF-I) infusion and dietary tri-iodothyronine (T3) supplementation on growth, body composition and plasma hormone levels in sex-linked dwarf mutant and normal chickens.

This study used a sex-linked dwarf mutant (SLD) chicken to evaluate growth-promoting and metabolic effects of recombinant human insulin-like growth factor-I (rhIGF-I) treatment. The SLD chicken is characterized by a 30% reduction in body weight and by high plasma GH levels, low plasma IGF-I and triodothyronine (T3) levels and very low GH binding on liver membranes, suggesting reduced functional GH receptors compared with normal chickens. The effects of a continuous s.c. infusion by osmotic mini-pump of 0.1 mg rhIGF-I/kg per day from 4 to 8 weeks of age on body weight, bone growth and body composition were investigated in female SLD and normal chicks. In addition, half of the birds received a dietary supplement of T3 (0.1 parts per million). Plasma levels of IGF-I, GH, T3, thyroxine and insulin were followed during the treatment. In normal chicks, rhIGF-I infusion had no effect on growth and little effect on plasma hormone levels except for a decrease in plasma insulin. In dwarf chicks, rhIGF-I infusion slightly increased body weight but had no effect on longitudinal bone growth. In addition, plasma GH levels were decreased and T3 levels remained lower than in normal chicks. Normal and dwarf chicks showed a decrease in abdominal fat after both IGF-I administration and T3 supplementation, the treatments having additive effects in dwarf chicks only. The combined rhIGF-I and T3 treatment restored a quasi-normal hormonal pattern in dwarf chicks, except for insulin which remained lower than in normal chicks. These results suggest that IGF-I in the chicken has no direct endocrine effect on statural growth.

The relationship of several genes suppressing plumage colour with body weight, food intake and feather loss of laying hens.

1. Growth rate, egg number, egg and adult body weight, plumage condition, food intake and efficiency of laying hens were compared in birds differing in plumage colour genotype at five loci (C, I, S, Ig, B). 2. Laying rates of the different genotypes were not different. 3. Growth rate and adult body weight of females were significantly less in loci C and I white females as compared with the corresponding coloured females; there was a similar tendency in adult weight for the other loci, and in egg weight. Differences in body weight were more marked in birds kept in cages than in those kept on the floor. 4. For all loci the genotype suppressing colour was associated with less plumage deterioration, this being highly significant for loci C and S. 5. Food intake, adjusted for body weight and egg production, was less in locus I white hens, in agreement with previous data concerning the C locus. 6. Pleiotropic effects associated with colour were suggested and their consequences on efficiency of egg production are discussed.