ACTA VETERINARIA ET ZOOTECHNICA SINICA ›› 2015, Vol. 46 ›› Issue (10): 1775-1783.doi: 10.11843/j.issn.0366-6964.2015.10.010

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Cryopreservation of the Chicken Blastodermal Cells Using the DMSO and Straw

WANG Kun1,2，ZHANG Bai-zhong1,YI Kang-le1,2，ZHU Li-jun1，JIANG Jun2，YAN Hai-feng1,3*   

  1. (1.Hunan Institute of Animal and Veterinary Science，Changsha 410131,China；2.College of Animal Science and Technology，Hunan Agricultural University，Changsha 410128，China;3.Hunan Tianxin Yellow Chicken Breeding Co.,Ltd.，Changsha 410143，China）
  • Received:2015-01-19 Online:2015-10-23 Published:2015-10-23

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Abstract:

For developing efficient cryopreservation technology of chicken blastodermal cells (BCs) and storing female poultry genetic resources，the BCs was frozen using dimethyl sulfoxide (DMSO) and straw，and the different DMSO concentration，freezing and thawing rate were compared by calculating the cell viability (CV) using fluorescein diacetate (FDA) and cell adherent rate cultivated for 24 h (CAR).Results showed that：(1) For the concentration of DMSO，the highest CV was in 20% group (0.58) and had no significant difference with group 15% (P>0.05)，but had extremely significant difference with group 5%，10% and 25% (P<0.01).The highest CAR was 30.5% in group 20%，and had no significant difference with group 15% (P>0.05)，but had significant difference with group 25% (P<0.05)，and had extremely significant with group 5%,10% (P<0.01).(2)The 3 steps to freeze BCs was used，and the first step was cooling at a rate of -1 ℃•min-1 to -7 ℃ and kept 10 min，the second step was to continue cooling the temperature at -15,-35,-55 or -75 ℃ respectively，the third step was to plunge the straws into liquid nitrogen.The highest CV was 0.53 of group -75 ℃，and had no significant difference (P>0.05) to group -35 or -55 ℃.The CAR had extremely significant difference within each groups (P<0.01)，and the highest was 36.6% in group -35 ℃.(3) The thawing rate at 37 ℃ water bath for 10 s，1 min，2 min and 3 min，the CV of each groups had no significant difference (P>0.05)，the highest one was 0.60 in 10 s group.The CAR was 43.9% in group 1 min，and it had no significant difference (P>0.05) with group 2 min，had significant difference (P<0.05) with the group 10 s and had extremely significant difference (P<0.01) with group 3 min.The thawing rate of 50 ℃ water bath for 5 s，20 s，40 s or 1 min，the highest CAR was 0.7 in group 5 s，and it had significant difference (P<0.05) with group 20 s，had extremely significant difference (P<0.01) with group 40 s and 1 min.The CAR thawed at 50 ℃ had extremely significant difference within the 4 groups (P<0.01).The highest CAR was 36.9% in group 5 s，and the lowest was 5.6% in group 1 min.In conclusion，the BCs frozen using 20% DMSO and straw，cooling at -1 ℃•min-1 to -7 ℃ for 10 min，continuing to cool at -35 ℃ at the same ratio and plunging into liquid nitrogen，then thawed by 37 ℃ water bath for 1-2 min，could get not only good CV，but also CAR.

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