Establishment of a cryopreservation system and evaluation of post-thaw effects for Piao chicken primordial germ cells

Abstract

Primordial germ cell (PGC) cryobanking provides an alternative strategy for conserving endangered chicken genetic resources; however, evidence regarding the post-thaw stability of breed-specific PGCs remains limited. This study evaluated a stepwise two-solution cryopreservation procedure based on CO₂-independent medium for gonad-derived Piao chicken PGCs stored for 3, 6, 9, and 12 months. Post-thaw characteristics were assessed by morphological observation, periodic acid–Schiff staining, trypan blue exclusion, EdU incorporation, CCK-8 analysis, JC-1 staining, quantitative reverse transcription PCR, immunofluorescence, flow cytometry, and embryonic gonadal homing analysis. Cryopreserved PGCs retained typical round or oval morphology and remained periodic acid–Schiff positive after thawing. Post-thaw mortality remained low (approximately 4–5%). No statistically significant differences were detected in mortality, proliferation-associated or metabolic parameters, or the expression of the evaluated genes (P > 0.05). JC-1 fluorescence patterns were qualitatively similar among the evaluated groups. DDX4 and SSEA-1 protein signals remained detectable after cryopreservation, and the embryo-level gonadal-homing-positive proportions were 70% in the Fresh group and 78% in the Frozen 12 M group, with no statistically significant difference between the two groups. These findings provide preliminary evidence that selected cellular and molecular characteristics were retained during the evaluated 12-month storage period. Larger biological sample sizes, quantitative assessment of donor-cell number and distribution within individual gonads, germline transmission testing, and genomic and epigenetic stability assessments are still required.