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Ultrastructural Characteristics of Fresh and Frozen-Thawed Ovine Embryos Using Two Cryoprotectants¹

^a Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria A, Dpt. de Reproducción Animal y Conservación de Recursos Zoogenéticos, 28040 Madrid, Spain ^b Consejo Superior de Investigaciones Científicas, Centro de Investigaciones Biológicas, 28006 Madrid, Spain ^c Servicio de Investigación Agroalimentaria, Dpt. de Tecnología en Producción Animal, 50080 Zaragoza, Spain

Cryopreservation of sheep embryos with ethylene glycol as a protectant appears to be more effective than glycerol, particularly at the morula stage, as has been demonstrated on the basis of in vitro and in vivo development rates after thawing. In this study we compare the ultrastructure of fresh morulae, thawed morulae, and blastocysts cryopreserved with either ethylene glycol or glycerol at the electron microscopic level, to look for cellular damage that could be responsible for proven differences in embryo survival after transfer. Embryos cryopreserved with glycerol showed unequal degrees of conservation even among blastomeres within a single embryo. In morulae, inner blastomeres were completely damaged, whereas external ones appeared to be intact. Both morulae and blastocysts cryopreserved with ethylene glycol showed a higher uniformity in blastomere conservation than embryos with glycerol. The most remarkable features in this experimental group were the presence of desmosomes following tight junctions between blastomeres and the presence of many microvilli on the outer surface of external blastomeres. These characteristics are similar in fresh embryos of the control group. Our results show that ethylene glycol protects membrane and cytoplasmic structures of embryonic cells from cryoinjury much better than glycerol. In vivo survival of embryos confirmed the ultrastructural observations. A limited permeability of glycerol would explain the observed ultrastructural differences in blastomere integrity, which depends on blastomere location and the differences between morulae and blastocysts. We conclude that the low reproductive yield after cryopreservation using glycerol can be attributed to the lack of protection of inner cells.

First decision: 10 July 2001.

¹ This work was supported by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (project SC97-020-C2).

² Correspondence: M.J. Cocero, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Dpt. de Reproducción Animal y Conservación de Recursos Zoogenéticos, Carretera de Ca Coruña km.5.9, 28040 Madrid, Spain. FAX: 34 915490956; cocero{at}inia.es