Viability and Functionality of Primordial Germ Cells after Freeze-thaw in Chickens

Abstract

The purpose of this study is to clarify the effects of freeze-thaw treatment on viability and functionality of primordial germ cells (PGCs) in chickens. PGCs were collected separately from embryonic blood of White Leghorn, Barred Plymouth Rock and Fayoumi breeds. Some PGCs were labeled with the fluorescent lipophilic carbocyanine dye to analyze functionality by transfer assay. 100 PGCs were used for each experiment to ensure accuracy of the test results. In the experimental group, PGCs were slow-frozen, then stored in liquid nitrogen for 1 month. In the unfrozen control group, PGCs were utilized immediately. The recovery rate of PGCs after freeze-thaw was 54.3%. The viability of PGCs in the frozen group was significantly lower than that of the control group (P<0.05) (85.7% vs. 99.2%) with no significant difference between the three breeds. Thirty fluorescent-labeled PGCs were randomly chosen from each set of recovered PGCs after freeze-thaw in the frozen group, and from each set of 100 PGCs in the control group, for transfer into the bloodstream. Gonadal migration of transferred PGCs was observed in all embryos in both test and control groups. The number of PGCs settled in the gonads of embryos at stage 27 was 52.8% lower in the frozen group than in the unfrozen control group (P<0.05). In the control group, significant difference in establishment of PGCs in stage 27 gonads was observed between the three breeds, with White Leghorn chickens harboring the most PGCs, and Barred Plymouth Rock chickens the fewest. We conclude that freeze-thaw treatment causes a decrease in functionality of PGCs in chicken, with only an estimated 46.5% of frozen PGCs being viable after thawing.