Fertile Silkie broiler eggs were obtained from the Silkie pedigree flock kept by Cobb-Europe at the Colchester United Kingdom facility. The Silkie flock are maintained under a scheduled feed regime (not ad libitum) and egg lay averages 138 eggs per hen. Silkie broiler eggs were incubated at 37.5∘C in a humidified incubator. A 1.0 μl blood sample was collected from the dorsal aorta of individual day 2.5 (stage 16 HH) Silkie embryos. After blood sampling a tissue sample from each embryo was frozen for sexing as determined by W chromosome PCR analysis (Clinton, 1994 (link)). The blood sample containing PGCs was cultured in FAOT medium as previously described (Whyte et al., 2015 (link)) for 4 weeks until reaching 50,000–100,000 PGCs. PGCs were cryopreserved in Stem-Cellbanker (Amsbio) in 3–4 aliquots until thawed for surrogate host injections. Once thawed PGCs were cultured for 1 week and the sex of the cultured PGCs was confirmed by a W chromosome PCR analysis (Clinton, 1994 (link)).
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