Viability of oocytes and granulosa cells from cryopreserved ovine ovarian primordial, primary and secondary follicles

Abstract

The study aimed to evaluate the effect of the addition of sucrose to the freezing and washing medium on the morphology and viability of ovine primordial, primary and secondary follicles and their enclosed oocytes and granulosa cells. Ovine primordial, primary and secondary ovarian follicles were cryopreserved in the absence or presence of 0.5M sucrose, with or without 1.0M ethylene glycol (EG) or 1.0M dimethyl sulphoxide (DMSO). After thawing, all follicles were washed in minimum essential medium (MEM), with or without 0.3M sucrose. Inclusion of sucrose in the freezing media generally improved the post-thaw morphology of the preantral follicles. The addition of sucrose to the washing media did not affect the percentage of normal follicles cryopreserved in the sucrose-containing media. However, its addition to the washing medium of ovarian tissue cryopreserved in the presence of EG, did increase the percentage of normal follicles. Although all the cryopreservation treatments lowered the percentage viability of the different isolated preantral follicle classes and their oocytes, the addition of sucrose to the cryoprotectants EG and DMSO appeared beneficial for the viability of all preantral follicles. The percentages recorded were lowest for the secondary follicles. In the presence of sucrose, cryopreservation of oocytes and granulosa cells from primordial and primary follicles was better than that from the cellular compartments of secondary follicles. It can thus be said that oocytes and granulosa cells of primordial and primary ovine follicles are well-cryopreserved in the presence of EG or DMSO, supplemented with sucrose, followed by a thawing-washing procedure in a sucrose-free medium.