Development of an allogenic in-ovo assay for primary chicken follicle stimulation

Here, we established an optimized co-culture for primordial and primary chicken follicles implanted into the chorioallantoic membrane of cultured eggs with growing embryos in surrogate shells (CAM-assay) for direct access during their initial growth and germline specification. The germline specification is initiated by germ plasm accumulation in the Balbiani body of early primordial follicles directed by the maternally provided germ plasm organizer Bucky ball. To investigate the regulatory network during this initial germline specification, we developed a protocol for the in-vitro lipofection of separated primordial and primary follicles with interfering RNAs. The implantation of lipofected follicles in the nourishing environment of the CAM of preincubated eggs and in-ovo culture of these stimulated follicles for further 2-4 days could be used to investigate alterations in the resulting transcription and protein profiles. To ensure optimized developmental conditions for the implanted follicles, either preincubated eggs fenestrated with a shell opening of about one square centimeter or preincubated eggs transferred in surrogate shells through an opening of 35 mm diameter, were compared as recipients. The development of the follicles in a three-dimensional culture system using surrogate shells ensures maximum access to the surface of the CAM and an exceptionally high vitality of the recipient embryos as well as the implanted follicles. Phenotypical parameters for the vitality assessment of the follicles after 2-4 days of co-incubation are provided. First data of interference effects with siRNAs for Bucky ball and CVH are added.