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United States Department of Agriculture

Agricultural Research Service

Title: A Method for Cryopreserving Chicken Primordial Germ Cells

Authors
item Moore, Daniel
item PURDY, PHIL
item BLACKBURN, HARVEY

Submitted to: Poultry Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 4, 2006
Publication Date: October 1, 2006
Citation: Moore, D.T., Purdy, P.H., Blackburn, H.D. 2006. A Method for Cryopreserving Chicken Primordial Germ Cells. Poultry Science. 85:1784–1790.

Interpretive Summary: This study established a method for preserving chicken primordial germ cells (PGCs) that enables long-term storage in liquid nitrogen. Gonads were harvested from stage 27 chick embryos and pooled in groups of 5 (5E), 10 (10E), or 20 (20E) embryos contributing gonads to the cell suspension. The cells were then frozen in one 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 frozen in a cryopreservation straw at a rate of -1ºC/minute until reaching -85ºC, and then plunged into liquid nitrogen, where they were stored until analysis. Flow cytometry was used to analyze the PGCs post-thaw with fluorescent probes and to determine viable PGC populations. The highest level of viable PGCs per individual embryo was observed for 10% EG with 10E, and was significantly higher than cryopreservation in 2.5% DMSO with 10E and 20E, 2.5% EG with 10E, 5% EG with 10E and all 0% cryoprotectant treatments. The highest percentage of viable PGCs was observed at 10% EG with 10E. It was demonstrated that PGCs were successfully frozen and the most effective treatment was 10% EG with 10 embryos/straw.

Technical Abstract: This study established a method for preserving chicken primordial germ cells (PGCs) that enables long-term storage in liquid nitrogen. Gonads were harvested from stage 27 chick embryos and pooled in groups of 5 (5E), 10 (10E), or 20 (20E) embryos contributing gonads to the cell suspension. The gonadal cells including PGCs were then frozen in one 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 -1ºC/minute until reaching -85ºC, and then plunged into liquid nitrogen (-196ºC), where they were stored until analysis. Flow cytometry was used to analyze the PGCs post-thaw. The PGC marker stage-specific embryonic antigen-1 (SSEA-1), which was detected with goat anti-mouse IgM fluorsecein isothiocyanate (FITC), was used to label all PGCs and propidium iodide (PI) was used to detect cells with compromised cell membranes. There was an interaction effect for the number of viable PGCs per individual embryo (P less than or equal to 0.05). The highest level (183.6 ± 28.4) of viable PGCs per individual embryo was observed for 10% EG with 10E, and was significantly higher (P less than or equal to 0.05) than cryopreservation in 2.5% DMSO with 10E and 20E, 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 PGCs. However, the highest percentage (80.6%) was observed at 10% EG with 10E. It was demonstrated that PGCs were successfully frozen and the most effective treatment was 10% EG with 10 embryos/straw.

Last Modified: 7/28/2014
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