Bovine oocyte membrane permeability and cryosurvival: effects of different cryoprotectants and calcium in the vitrification media

The cryopreservation process must be improved to enhance oocyte cryosurvival and functionality. Two protocols with different cryoprotectants (CPAs), containing either ethylene glycol (EG), dimethyl sulfoxide (DMSO) and sucrose (EGDMSO) or 1,2-propanediol and sucrose (PrOH) were evaluated. In both protocols, calcium (Ca²⁺) free or -containing base media were tested. Oocytes were subjected to vitrification or only exposed to CPAs without immersion in liquid nitrogen. Oocyte's viability, cortical granules location and competence for development after fertilization were assessed. Finally, fatty acid composition and membrane permeability of oocytes exposed to CPAs were analyzed. Independently of Ca²⁺ concentration in the vitrification media, the development rates were higher in oocytes vitrified with EGDMSO protocols (p = 0.0005). After warming, higher cleavage rates were obtained in EGDMSO + Ca²⁺ compared to the PrOH without Ca²⁺ protocol (p = 0.02). Oocytes exposed to PrOH without Ca²⁺ presented lower cleavage rates compared to control (p = 0.04). An enhanced premature zona hardening in vitrified oocytes as well as lower concentrations of the fatty acids c11:18:1 and 20:4n-6 in cumulus oocyte complexes exposed to PrOH protocols were identified. The oocytes minimum volume and permeability were affected by the exposure to PrOH and Ca²⁺ (p ≤ 0.007). In conclusion, the most effective protocol for bovine oocytes cryopreservation combines EG and DMSO, independently of Ca²⁺ concentration in the media. A higher toxicity and an incomplete depletion of water during PrOH loading may hamper oocyte viability. The type of CPAs and Ca²⁺ interfered differentially on oocyte pathways to functionality, and this should be considered when choosing a cryopreservation protocol.