Survival of Vitrified Water Buffalo Cumulus Oocyte Complexes and Their Subsequent Development in Vitro

The study aimed to determine the effects of different concentrations of glycerol and ethylene glycol and ultrarapid cryopreservation (vitrification) on survival and subsequent in vitro development of bubaline cumulus oocyte complexes (COCs) in order to recognize the optimum cryoprotectant. Survival and in vitro maturation, fertilization and cleavage of buffalo COCs was evaluated subsequent to their cryopreservation by vitrification. The vitrification solution (VS) consisted of Dulbecco’s phosphate buffered saline (DPBS) supplemented with 0.5 M sucrose, 0.5% bovine serum albumin (BSA) and different molar (M) concentrations of the cryoprotectants glycerol (G) (4 M, 6 M, 8 M and 10 M), ethylene glycol (EG) (4 M, 6 M, 8 M and 10 M) and their combinations (2 M G + 2 M EG, 3 M G + 3 M EG, 4 M G + 4 M EG and 5 M G + 5 M EG). The COCs were pre-equilibrated in 50% of the VS for 3−5 min, then kept in VS for 1 min and loaded in pre-sterilized 0.25 mL semen straws. After 7−10 days of storage COCs were warmed (38 C for 5 s) and evaluated for morphological damage. Morphologically normal COCs were cultured in vitro and evaluated for nuclear maturation (n=847), fertilization (n=621) and cleavage (n=1451) in two separate experiments. The survival of oocytes was 86.4% and 89.6% in experiment 1 and 2. The highest proportion of normal oocytes was seen in 6 M EG and the lowest − in 10 M G in both experiments. The in vitro maturation of oocytes at the end of experiment 1, and the in vitro fertilization and cleavage at the end of experiment 2, were significantly lower in all tested vitrification cryoprotectants compared to control. A dose-dependant increase in the proportion of oocytes matured, fertilized or cleaved was seen for both G and EG up to concentrations of 8 M. There was no specific benefit of combining G and EG on the subsequent in vitro maturation, fertilization and cleavage of oocytes. At equal concentrations EG proved to be a better cryoprotectant than G. It was concluded that there appeared to be a limit to the concentration of the cryoprotectant (8 M) beyond which the cryoprotectant exerted suboptimal effects and that there was no benefit of combining two similar cryoprotectants for cryopreservation of oocytes by vitrification.