Cryopreservation of zebrafish (Danio rerio) blastomeres by controlled slow cooling

Cryopreservation is now a common practice in the fields of aquaculture, conservation and biomedicine. One approach for maintaining the genetic diversity of both nuclear genome and mitochondrial DNA is cryopreservation of the blastomere. This study sets out to determine an optimum cryopreservation protocol for blastomeres isolated from 50% epiboly zebrafish embryos. Freezing was performed in 0.25 ml straws in a programmable freezer. The optimum slow cooling protocol is identified as 5 degree C per min, from 22 to -6 degree C, holding for 15 min, 0.3 degree C per min from -6 to -40 degree C, 2 degree C per min from -40 to -80 degree C, cells were held for 10 min at -80 degree C before plunging into the liquid nitrogen. Thawing was performed in a water bath at 28 degrees C for 15 s followed by four step-wise removal of the cryoprotectant. Blastomeres had the highest survival level (70.2 +/- 3.2%) when a mixture of 1.5 M dimethyl sulfoxide (DMSO) and 0.1 M sucrose were used as cryoprotectants. This was higher than that achieved with either of the sugar alternatives, trehalose (60.6 +/- 3.1%) or glucose (43.1 +/- 4.9%). In the present study, cryopreservation of 50 percent epiboly zebrafish blastomeres was studied for the first time using controlled slow cooling method.