Evaluation of non-penetrating sugar cryoprotectants and antioxidants during chicken blastodermal cell cryopreservation

Chicken egg blastodermal cell (BC) cryopreservation is an effective strategy for conservation of complete genome of a breed. The present study evaluated non-penetrating sugars sucrose, trehalose and raffinose, and antioxidants tempol, betaine and ascorbic acid at different concentrations during Kadaknath chicken BC cryopreservation. BCs isolated from fresh Kadaknath eggs were cryo-preserved in DMEM containing 10% dimethylsulfoxide (DMSO) and 10% FBS. In independent trials non-penetrating sugars sucrose (0, 0.1, 0.2 M), trehalose (0, 0.1, 0.2, 0.4 M) and raffinose (0, 0.1, 0.2 M), and tempol (0, 1, 5 mM), betaine (0, 0.1, 0.2, 0.4 M), ascorbic acid (0, 25, 50, 100 µM) were added to the cryopreservation mixture and evaluated. The medium containing BCs was filled in 0.25 mL plastic straws and exposed to liquid nitrogen vapours for 30 min. The straws were then plunged into liquid nitrogen and stored at least for fifteen days before thawing at 20 oC for 20 seconds and evaluation. The post-thaw percentage of live cells was assessed using 0.4% trypan blue staining. Both evaluated concentrations of sucrose significantly (P<0.05) increased the post-thaw live cell percentage. Betaine at 0.2 M concentration increased whereas ascorbic acid at 100 µM concentration decreased the post-thaw live cells. Trehalose, raffinose and tempol did not affect the post thaw BC live cells percentage. In conclusion, the results demonstrate that addition of sucrose and betaine during blastodermal cell cryopreservation improves the post thaw live cells.