Parent Stock: Vehicle and CORT Treatments

The CORT treatment aimed to simulate eggs that received an increased concentration of corticosterone from a stressed hen. The concentration of corticosterone in egg yolks has been previously reported to range from 0.77 to 2.8 ng/g in Hy-Line Brown (Navara and Pinson, 2010, Ahmed et al., 2014, Engel et al., 2019) to average 1.6 ng/g in Hy-Line White (Navara and Pinson, 2010) and 2.13 ng/g in Bovan White (Haussmann et al., 2012) under control conditions. However, analytical validation of enzyme immunoassay and radioimmunoassay techniques showed the presence of cross-reactive substances that hamper quantification of corticosterone in the yolk and albumen of eggs (Rettenbacher et al., 2013a). Because the exact concentration of corticosterone in eggs remains unknown, we followed the methodology and dosage developed by Janczak et al. (2007a) and subsequently used by Haussmann et al. (2012). The CORT treatment group received a final concentration of 10 ng of corticosterone/mL of egg content (corticosterone: Sigma-Aldrich Chemical Co., St. Louis, MO) diluted in sesame oil (Fisher Scientific Co., Fair Lawn, NJ), while the vehicle treatment group received the same concentration of sesame oil only. The average weight of egg content (90% of egg weight) (Beuving and Vonder, 1981) per offspring group was 50, 50, and 59 g. Thus, 50 μL of solution was injected into eggs from breeders of 32 and 52 wk of age, whereas 60 μL was injected into eggs from 72-wk-old hens.

When breeders were at 25 wk of age, we collected eggs from all strains and conducted a pilot study to estimate hatchability levels. Eggs used to form the different offspring groups were selected according to weight (between 52 and 70 g) and day of laying (recent over old). One day before incubation, a 5 x 5 mm layer of silicone sealant (General Electric, Boston, MA) was applied onto the basal tip of the eggs designed to form the injected treatments to prevent gas exchange and contamination. On the morning of each incubation day, a stock solution of 2.5 mg corticosterone was diluted in 2.5 mL sesame oil, warmed to 100°C, and sonicated for 15 min. Both CORT and vehicle solutions were sterilized at 180°C for 30 min and let to cool down to room temperature. Moments before incubation, 1-ml syringes were filled, and treatments were injected 5 mm into the albumen through the silicone seal using 23-gauge needles.