A comparative study of vitrification and slow freezing on subsequent developmental capacity of immature sheep oocytes

The present study evaluated the post warming morphologic survival, and subsequent in vitro maturation and development of immature ovine oocytes cryopreserved by either conventional slow freezing or vitrification. Cumulus oocyte complexes (COC's, n=300) retrieved from slaughter house ovaries were serially kept for 10 min each in a freezing solution (FS) containing different concentrations of glycerol (3.3%, 6.6% and 10%) prepared in Modified dulbeccos phosphate buffered saline supplemented with 10.0% fetal calf serum, 10% sucrose and 1.0% antibiotic-antimycotics. COCs (10–15) were then loaded in pre-sterilized 0.25 mL empty semen straws and cryopreserved by either conventional slow freezing (n=150) or vitrification (n=150). After 7–10 days of cryostorage in liquid nitrogen, COCs were warmed and evaluated for morphologic damage. Morphologically normal COCs were matured in vitro in TCM-199, evaluated for cumulus expansion and fertilized with frozen ram semen capacitated in Brackett and Oliphant medium supplemented with heparin and caffeine. Inseminated oocytes were cultured in vitro for 5–6 days for embryo development. Freshly collected oocytes (n=100) were also matured, fertilized and cultured in vitro and kept as control. A significantly (p<0.05) higher proportion of morphologically normal oocytes were recovered after vitrification-thawing than those obtained with slow cooling (73.33 vs 56.67%, respectively). Among the damaged oocytes, cracking of zona pellucida and leakage of cellular contents were the most frequently observed abnormalities. A significantly higher (p<0.05) proportion of in vitro maturation was observed for vitrified-warmed oocytes in glycerol than those obtained with slow freezing. A similar trend was observed for development to morula and blastocyst stage. However, in vitro developmental competence was higher for fresh oocytes (control) than those obtained for vitrified or slow frozen oocytes. In conclusion, vitrification of sheep oocytes in 10% of glycerol yields better oocyte survival, in vitro maturation and embryo development compared to conventional slow freezing.