Embryonic Injections

Fertilized eggs of domestic chicks (Gallus gallus) of the Ross 308 (Aviagen) strain were obtained from a local commercial hatchery (Agricola Berica, Montegalda, Italy) and incubated at 37.7°C and 60% of relative humidity in the darkness. The first day of incubation was considered embryonic day 0 (E0). At E14, fertilized eggs were selected by candling before injection. Embryo injection was performed according to previous reports (Nishigori et al., 2013; Sgadò et al., 2018). Briefly, a small hole was made on the eggshell above the air sac, and 35 μmoles of VPA (Sodium Valproate, Sigma-Aldrich) dissolved in double distilled injectable water were administered to each fertilized egg, in a volume of 200 μl. Age-matched control eggs were injected using the same procedure with 200 μl of vehicle (double distilled injectable water). After sealing the hole with paper tape, eggs were placed back in the incubator (FIEM srl, Italy). Previous reports have analyzed the effect of different doses and time of administration of VPA on embryonic development in different vertebrate species (see for a review Roullet et al., 2013; Ranger and Ellenbroek, 2016). The typical dose and time of administration in rodents is 200–500 mg/kg in acute, single dose administration between E12 and E14. In domestic chicks, administration of 35 μmoles/egg (corresponding to approximately 100 mg/kg) has been tested between E10 and E14 with differential effects on hatching rate, showing a dramatic decrease of hatchings at E10 and a significant decrease of hatchings at E12 but no significant effect at E14 (Nishigori et al., 2013). Administration of 35 μmoles/egg at E14 induced social deficits without affecting hatchability, motor behavior and imprinting abilities (Nishigori et al., 2013; Sgadò et al., 2018).

During incubation and hatching, eggs and chicks were maintained in complete darkness, preventing any visual experience prior to the test. Controlling the visual experience during pre- and post-natal development enable to exclude any interference of visual stimuli in the expression of predispositions toward animacy cues, and to demonstrate the innate nature of these mechanisms. Each chick was tested only once.