Effects of cryopreservation procedures on the cytology and fertilization rate of in vitro-matured bovine oocytes

The survival and developmental capacity of bovine oocytes after cryopreserv ation are greatly impaired, possibly due to organelle damage caused by free zing procedures. Distributions of chromosomes, microtubules, and microfilam ents in bovine oocytes matured in vitro were examined after cooling, ethyle ne glycol (EG) exposure, or freezing. Oocytes were incubated after treatmen t for 20 min or 1 or 3 h, fixed, and evaluated using specific fluorescent p robes. Abnormal cytological features increased over control levels after co oling or EC exposure and rewarming. Changes observed in oocytes during pref reezing manipulations included chromosome dispersal and clumping, microtubu le depolymerization and alteration of spindle structure, and formation of c raters 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 po stthaw (versus 90% of controls). Only 7-14% of frozen-thawed oocytes had no rmal spindles (versus 59-71% of controls). Normal distribution of filamento us actin was observed in less than 30% of oocytes postthaw (versus 62-89% o f controls). These results indicate that the steps in a conventional freezi ng procedure cause irreversible alterations in multiple cytological compone nts of bovine oocytes, demonstrating the need for improved strategies for p reventing cellular damage during cryopreservation procedures.