The Effect of Simple Freezing Method on Viability of Frozen-thawed Primordial Germ Cells on the Chicken

동결 닭 PGCs의 생식계열 키메라를 이용한 생체에의 복원을 실용화 하기 위해서는, 닭 PGCs의 동결 보존 기술의 향상에 의해 동결 및 융해 후의 많은 생존 세포를 확보하는 것과, 생식계열 키메라의 제작 효율을 높이는 것이 반드시 필요하다. 닭 PGCs는 배양 5.5~6일령의 닭 원시 생식선으로부터 채취하고, MACS 방법에 의해서 순수 닭 PGCs를 분리했다. 15% 각각의 EG와 DMSO를 동결 보호제로 사용한 처리군이 각 군의 농도에 상관없이 유의적(p<0.05)으로 PG 처리군보다 동결 및 융해 후의 세포의 생존율이 높음을 확인하였다. 특히, 10% EG+FBS 조합의 처리군에서 상업용 닭인 한협육종협회3호종(B : 88.7%)이 오계종(A : 85.1%), 아사 브라운종(C : 84.6%) 그리고 화이트 레그혼종(D : 85.9%)의 세 품종보다 동결 및 융해 후의 닭 PGCs의 생존율이 유의적(p<0.05)으로 높음을 확인하였다. 간이 동결에 있어서 가장 높은 생존율을 보인 10% EG이 10% DMSO와 함께 최적의 동결 보호제로서 사용 가능성을 확인하였다. This study was conducted to establish the method for preserving chicken primordial germ cells (PGCs) that enables long-term storage in liquid nitrogen ( $LN_2$ ) for developmental engineering or preservation of species. The purpose of this study is to clarify the effects of simple freeze-thaw treatment on viability of PGCs in chickens and to the optimal protocol for PGCs freezing. PGCs obtained from the germinal gonade of an early embryos of 5.5~6 day (stage 28) of Isa Brown, Korean Ogye (KO), White Leghorn and Commercial breeds, using the MACS method were suspended in a freezing medium containing a freezing and protecting agents (e.g. dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG)). The gonadal cells, including PGCs, were then frozen in 1 of the following cryoprotectant treatments : 2.5%, 5%, 10%, 15%, and 0% cryoprotectant (DMSO, EG, PG) as a control. Effects of exposure to simple freezing, with different concentrations of the cryoprotectant solution, were examined. After simple freezing, the viability of PGCs after freeze-thawing was significantly higher for Commercial breeds ( $88.7{\pm}2.4%$ ) than KO ( $85.1{\pm}0.4%$ ), Isa Brown ( $84.6{\pm}0.2%$ ) and White Leghorn ( $85.9{\pm}0.1%$ ) (p<0.05) using 10% EG cryoprotectant. Therefore, these systems may contribute in the improvement of cryopreservation for a scarce species in birds preservation. This study established a method for preserving chicken PGCs that enables systematic storage and labeling of cryopreserved PGCs in liquid ( $LN_2$ ) at a germplasm repository and ease of entry into a database.