进行语法修改：22 Isolation and culture of PGCs in gonadsFreshly laid eggs from Gaoyou yellow chickens and Shouguang black chickens were incubated at 37°C for 55 days to obtain chicken embryos at Hamburger an

Isolation and Culture of PGCs in Gonads

To obtain chicken embryos at Hamburger and Hamilton (HH) stages 27-28, freshly laid eggs from Gaoyou yellow chickens and Shouguang black chickens were incubated at 37°C for 5.5 days. Individual gonads were isolated using fine forceps under a dissecting microscope and washed in 300 μl of phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA). After centrifuging and removing the supernatant, the gonad tissue was incubated in 200 μl of 0.25% trypsin-EDTA for 1 minute and then mechanically dissociated into a suspension using a pipette. The digestion was stopped by adding 300 μl of DMEM culture medium containing 5% fetal bovine serum (FBS), and the suspension was centrifuged and the supernatant was removed. The cell pellet was resuspended in FAcS culture medium and transferred to a 48-well culture plate for cultivation. The medium was changed every 3 days by replacing 1/3 to 1/2 of the medium, until the cell number reached 1×10^6 for cryopreservation.

The chicken PGC culture medium was prepared according to Whyte et al. (2015). The FAcS culture medium comprised DMEM (high glucose, no glutamine, no calcium) (Gibco, 21068028) as the base medium, supplemented with H2O (Sigma, W3500), 0.15 mM CaCl2 (100x) (Sigma, C7902), 1x B-27® Supplement (50x) (Gibco, 17504044), 2.0 mM GlutaMAXTM-1 (1000x) (Gibco, 35050061), 1x MEM NEAA (100x) (Gibco, 11140050), 0.1 mM 2-mercaptoethanol (55 mM) (Gibco, 21985023), 0.20% chicken serum (Gibco, 16110082), 1x EmbryoMax® Nucleosides (100x) (Sigma, E8-008-D), 1.2 mM sodium pyruvate (100 mM) (Sigma, 11360070), 0.20% ovalbumin (Sigma, A5503), 0.01% sodium heparin (1000x) (MCE, HY-17567A), 1x FGF-2 (2500x) (MCE, K128N), and 25 ng/ml human activin A (MCE, HEK293)