4.6. Chicken Intestinal Explant Culture

A 10 cm segment of the jejunum was collected from 1- to 2-week-old broiler chickens, and jejunal explants were prepared immediately after animals were sacrificed, as previously described [25,58]. In brief, after thorough washes of a jejunal segment in cold PBS supplemented with 100 µg/mL of gentamicin, 100 U/mL penicillin, and 100 µg/mL streptomycin, smaller segments (approximately 5 mm × 5 mm) were prepared and placed individually in 12-well plates containing 2 mL RPMI 1640 medium containing 10% FBS, 20 mM HEPES, 100 µg/mL gentamicin, 100 U/mL penicillin, and 100 µg/mL streptomycin; this was followed by the addition of each compound, in triplicate, at different concentrations. The explants were incubated at 37 °C for 24 h in a hypoxia chamber (StemCell Technologies, Vancouver, BC, Canada) flushed with 95% O₂ and 5% CO₂. Total RNA isolation and RT-qPCR analysis of chicken HDP gene expression were performed with jejunal explants after stimulation.