Identification of novel candidate genes for regulation of follicle selection in the avian ovary
McDerment, Neil Alastair
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Selective breeding of chickens for high growth rate and other production traits has led to the modern commercial broiler, a bird that has the genetic potential for reaching an average body weight of 2.7kg within 6 weeks of hatch. However, the breeding stock for modern broilers has to be feed controlled in order to lay large numbers of viable hatching eggs. Broiler breeders, when fed ad libitum, have a propensity to produce internal ovulations, double-yolked, misshapen or shell-less eggs. This is due to the release of multiple ova at ovulation, which results in a significant loss of production. Feed control has been shown to mitigate this effect but welfare concerns have been raised as to the side-effects for the birds. The main objective of this research was to determine the genetic basis for the regulation of ovarian follicle selection and its dysfunction in ad libitum-fed broiler breeders, and how this might be addressed by genetic selection to limit the impact on the management and welfare of future broiler breeders. A multi-layered statistical, expression profiling and cluster analysis of ovarian gene expression data from a microarray study was carried out to identify candidate genes for further study.Key stages of development were investigated for feed restricted and ad libitum-fed broiler breeders. Several gene candidate genes were validated by qPCR in a comparison of different ovarian tissues in layer type hens for subsequent analysis in broiler breeders. Sequencing of the founders of an Advanced Intercross Line (AIL) of commercial broiler breeders and White Leghorn layers was performed covering 3 regions of each of the primary candidate genes in order to identify genetic variation that could account for differences in follicle number between broilers and layers. Expression data from a microarray study highlighted a number of potential candidate genes for regulation of follicle development. One of these genes, Platelet Derived Growth Factor Receptor Like (PDGFRL), shares significant sequence homology with the active domains of Platelet Derived Growth Factor Receptor β. Expression profiling in layers showed peak PDGFRL expression in 5-6 mm follicles and the F2 follicle (P <0.001). PDGFRL was also up-regulated in response to ad libitum feeding in broiler breeders in 6-8 mm follicles (P<0.016), the point at which follicle selection and recruitment is considered to occur. In addition to this, while PDGFRL expression remains relatively constant between tissues under ad libitum conditions, it shows a clear reduction in expression (P <0.001) in prehierarchical follicles relative to the stroma and the F1 follicle under feed restriction. This observation is consistent with results from the original microarray study. Sequencing of the AIL Founders highlighted several SNPs in the broiler that have the potential to be used as markers for incorporation into commercial selection programs. EST alignment in preparation for targeted sequencing of PDGFRL also highlighted three potential forms of the protein, each with a different 5’ starting sequence. Initial investigation has shown all three to be expressed in ovarian follicles. QPCR in a panel of 13 tissues shows marked differences between the 3 variants, implying different and perhaps specialised roles for each. The PDGFR family has a potential role in steroidogenesis, and the expression profiling, combined with the clear effect on expression from ad libitum feeding in broiler breeders, suggest that PDGFRL is a strong candidate for involvement in the regulation of follicle development GDF9, shown to be associated with multiple ovulation in sheep, and FSH receptor, a mediator of neuroendocrine signalling to the ovary, were also investigated. They behaved as expected in layer type birds but both showed significant differential expression (P = 0.005 and 0.018 respectively) as a result of ad libitum feeding in broiler breeders. Though these two genes have been extensively investigated, these are previously unobserved effects. SNPs have also been identified in these genes which have the potential to be used as markers for incorporation into commercial selection programs. To fully exploit these results, additional investigation is recommended to confirm these results in commercial populations and to determine how they can be employed to best effect.