IN-VITRO MATURATION AND TRANSMISSION ELECTRON-MICROSCOPIC OBSERVATIONOF HORSE OOCYTES AFTER VITRIFICATION

This study was designed to examine the suitability of immature horse o ocytes for vitrification. Immature oocytes derived from slaughtered ho rse ovaries were transferred to a vitrification solution (EFS; 40% eth ylene glycol, 18% Ficoll, and 0.3 M sucrose in modified phosphate-buff ered saline) directly (Groups 1 and 4) or were first exposed to 20% et hylene glycol solution for 10 min (Groups 2 and 5) or 20 min (Groups 3 and 6). Oocytes were handled at 20 degrees C (Groups 1, 2, and 3) or 30 degrees C (Groups 4, 5, and 6). After vitrification and warming, th eir viability was assessed by maturation culture for 32 h. The percent ages of oocytes reaching the metaphase II stage after the in vitro mat uration in Groups 2, 3, 5, and 6 (16.0, 16.7, 10.0, and 8.2%, respecti vely) were higher than those in Groups 1 and 4 (2.2 and 1.9%, respecti vely). In untreated control oocytes, 55.6% completed meiosis in vitro. Transmission electron microscopy was used to compare the fine structu re of vitrified oocytes (treated as Group 2) with those of untreated c ontrol oocytes and EFS-exposed, nonvitrified oocytes (n = 10 each). Th e viability of EFS-exposed oocytes, assessed by in vitro maturation, w as 27.7%. Vitrification induced some ultrastructural changes, such as the swelling of mitochondria together with reduced matrix density, the destruction of communication between oocytes and their surrounding cu mulus cells, and the presence of vacuoles located in the periphery of the ooplasm. However, these changes were not always observed. Exposure of the oocytes to EFS solution induced similar ultrastructural change s in mitochondria and cell-cell communication but to a lesser extent. However, the exposure to EFS induced vacuoles in the periphery of the ooplasm to the same extent as did the vitrification. Thus, immature ho rse oocytes can be cryopreserved by vitrification with EFS solution. R educed viability of EFS-exposed and/or vitrified horse oocytes may rel ate to morphological changes such as destruction of the intercellular communications between cumulus cells and oocytes. (C) 1996 Academic Pr ess, Inc.