Effects of Cryopreservation Procedures on the Cytology and Fertilization Rate of In Vitro-Matured Bovine Oocytes1

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Abstract
The survival and developmental capacity of bovine oocytes after cryopreservation are greatly impaired, possibly due to organelle damage caused by freezing procedures. Distributions of chromosomes, microtubules, and microfilaments in bovine oocytes matured in vitro were examined after cooling, ethylene glycol (EG) exposure, or freezing. Oocytes were incubated after treatment for 20 min or 1 or 3 h, fixed, and evaluated using specific fluorescent probes. Abnormal cytological features increased over control levels after cooling or EG exposure and rewarming. Changes observed in oocytes during prefreezing manipulations included chromosome dispersal and clumping, microtubule depolymerization and alteration of spindle structure, and formation of craters and discontinuity in cytoskeletal actin staining. Freezing also led to an increase in the occurrence of cytological abnormalities. Less than 31% of frozen-thawed oocytes contained a normal chromosome arrangement 3 h postthaw (versus 90% of controls). Only 7-14% of frozen-thawed oocytes had normal spindles (versus 59-71% of controls). Normal distribution of filamentous actin was observed in less than 30% of oocytes postthaw (versus 62-89% of controls). These results indicate that the steps in a conventional freezing procedure cause irreversible alterations in multiple cytological components of bovine oocytes, demonstrating the need for improved strategies for preventing cellular damage during cryopreservation procedures.