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A Method for Cryopreserving Chicken Primordial Germ Cells¹

National Animal Germplasm Program, National Center for Genetic Resources Preservation, Fort Collins, CO 80521

² Corresponding author: Harvey.Blackburn{at}ars.usda.gov

This study established a method for preserving chicken primordial germ cells (PGC) that enables long-term storage in liquid N. Gonads were harvested from stage 27 chick embryos and pooled in groups of 5, 10 (10E), or 20 embryos, contributing gonads to the cell suspension. The gonadal cells, including PGC, were then frozen in 1 of the following cryoprotectant treatments: 2.5% dimethyl sulfoxide (DMSO), 5% DMSO, 10% DMSO, 2.5% ethylene glycol (EG), 5% EG, 10% EG, and 0% cryoprotectant as a control. The cells were liberated and frozen in a biosecure cryopreservation straw at a rate of –1°C/min until reaching –85°C and were then plunged into liquid N (–196°C), in which they were stored until analysis. Flow cytometry was used to analyze the PGC post-thaw. The PGC marker stage-specific embryonic antigen-1, which was detected with goat antimouse IgM fluorescein isothiocyanate, was used to label all PGC, and propidium iodide was used to detect cells with compromised cell membranes. There was an interaction effect for the number of viable PGC per individual embryo (P 0.05). The highest level (183.6 ± 28.4) of viable PGC per individual embryo was observed for 10% EG with 10E and was significantly higher (P 0.05) than cryopreservation in 2.5% DMSO with 10E and 20 embryos, 2.5% EG with 10E, 5% EG with 10E, and all 0% cryoprotectant treatments. No statistical interaction (P > 0.05) was observed for the percentage of viable PGC. However, the highest percentage (80.6%) was observed at 10% EG with 10E. It was demonstrated that PGC were successfully frozen, and the most effective treatment was 10% EG with 10 embryos/straw.

Key Words: primordial germ cell • cryopreservation • flow cytometry

¹ Mention of a trade name or proprietary product does not constitute a guaranty or warranty by the USDA and does not imply approval to the exclusion of others not mentioned. The USDA, Agricultural Research Service, Northern Plains Area, Fort Collins, CO, is an equal opportunity and affirmative action employer. All agency services are available without discrimination.