Vitrification of immature bovine oocytes in protein-free media: The impact of the cryoprotectant treatment protocol, base medium, and ovary storage

Abstract

Protein-free media are essential for the sanitary cryopreservation of bovine genetic resources. Our aim was to set up an optimized protocol for the vitrification of immature bovine oocytes using protein free media which can provide the highest embryo development rates and embryo quality after subsequent in vitro maturation and fertilization. First, using a protein free NCSU-37 as base medium we compared the efficacy of vitrification on Cryotop device with two different CPA protocols. “Protocol A″ employed a combination of ethylene glycol and propylene glycol as permeating cryoprotectants (pCPA) and equilibration in 4% total pCPA (2% ethylene glycol + 2% propylene glycol). “Protocol B″ employed a combination of ethylene glycol and DMSO and equilibration in 15% total pCPA (7.5% ethylene glycol + 7.5% DMSO). The 2 protocols were equally effective in terms of oocyte survival and subsequent development to the blastocyst stage. However, blastocyst cell numbers were significantly higher with “Protocol A”. TCM-199 and NCSU-37 were equally effective as base media for vitrification. Vitrification with “Protocol A″ reduced the percentage of live oocytes and subsequent development to blastocyst stage but did not affect the hatching and cell numbers of blastocysts when compared to the non-treated group. CPA treatment of “Protocol A″ without cooling did not affect embryo development. Storage of ovaries in PBS at 15 °C for overnight reduced the percentage of surviving oocytes after vitrification but not their subsequent development to the blastocyst stage. In conclusion we established a vitrification protocol for the cryopreservation of immature bovine oocytes employing protein-free media which provided high blastocyst quality without noticeable toxic effects.