In vitro and in vivo survival of cryopreserved sheep embryos

Experiments were conducted to devise an efficient method to cryopreserve ovine embryos for field application. Embryos were surgically collected from superovulated ewes on Day 6 after natural breeding; oocytes were collected from ovaries obtained at the abattoir. Osmotic behavior of oocytes and embryos was determined by measuring their responses to hypertonic solutions of CsCl or sucrose. Embryos and oocytes contracted osmotically by decreasing their volumes proportionally to the reciprocal of the solution's osmolality. The respective nonosmotic volumes of embryos in CsCl and sucrose were 13.8 and 13.5% of their isotonic volume, and those of oocytes were 18.5 and 19.6%. Tests of the permeability of morulae to commonly used cryoprotectants, ethylene glycol (EG), propylene glycol (PG), dimethyl sulfoxide (DMSO), and glycerol (Glyc), showed that the order of permeability was EG > PG > DMSO approximately Glyc. Comparison of the efficacy of cryoprotective agents indicated that the respective survivals of embryos frozen in EG, PG, and DMSO were 76.9, 62.5, and 55.6%, based on their development into hatched blastocysts in vitro. Therefore, EG appeared to be superior to the other two cryoprotectants for freezing sheep embryos. To determine the functional survival of embryos in vivo, 67 embryos frozen in EG were thawed and directly diluted with phosphate-buffered saline; 47 of these (70%) appeared morphologically normal and were transferred into 14 recipients. Five of these recipients, which had received a total of 16 embryos, became pregnant. Ten lambs were born, showing that the method employed in this study for cryopreservation of sheep embryos followed by their direct dilution out of EG has potential application for practical field use.