IN-VITRO AND IN-VIVO SURVIVAL OF CRYOPRESERVED SHEEP EMBRYOS

Experiments were conducted to devise an efficient method to cryopreser ve ovine embryos for field application. Embryos were surgically collec ted from superovulated ewes on Day 6 after natural breeding; oocytes w ere 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 contracte d osmotically by decreasing their volumes proportionally to the recipr ocal of the solution's osmolality. The respective nonosmotic volumes o f embryos in CsCl and sucrose were 13.8 and 13.5% of their isotonic vo lume, and those of oocytes were 18.5 and 19.6%. Tests of the permeabil ity 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 approximate to Glyc. Comparison of the efficacy of cryoprotective agents indicate d 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 bl astocysts in vitro. Therefore, EG appeared to be superior to the other two cryoprotectants for freezing sheep embryos. To determine the func tional survival of embryos in vivo, 67 embryos frozen in EG were thawe d and directly diluted with phosphate-buffered saline; 47 of these (70 %) appeared morphologically normal and were transferred into 14 recipi ents. Five of these recipients, which had received a total of 16 embry os, became pregnant. Ten lambs were born, showing that the method empl oyed in this study for cryopreservation of sheep embryos followed by t heir direct dilution out of EG has potential application for practical field use. (C) 1995 Academic Press, Inc.