Birth of calves after direct transfer of thawed bovine embryos stored frozen in ethylene glycol

Abstract

The present study investigated the use of ethylene glycol (EG) as a cryoprotectant before the direct transfer of frozen-thawed bovine embryos. Embryos at the morula to blastocyst stages collected on Days 6–8 (estrus designated Day 0) in phosphate buffered saline+20% newborn calf serum were placed into 1.8 M EG or in 1.8 MEG+0.25 M sucrose (EG+SUC), and equilibrated for 13–93 min at room temperature (20–25°C) or 37°C. Each embryo was then loaded into a 0.25 ml straw, and directly placed in the cooling chamber of a freezer precooled at −7°C. After 2 min at −7°C, the samples were seeded, then held for a further 8 min at −7°C, and cooled to −25 or −30°C at −0.3°C min −1 before being plunged into liquid nitrogen. Control embryos were also frozen with 1.4 M glycerol +0.25 M sucrose (GLY+SUC). Frozen embryos were thawed in a water bath at 37°C (EG and EG+SUC) or 20°C (GLY+SUC). After thawing the straws containing an embryo frozen in EG, EG+SUC or GLY+SUC were directly mounted into an embryo transfer gun and transferred to recipients without diluting of the cryoprotectants. The pregnancy rates were 69% ( 20 29 ), 52% ( 13 25 ) and 60% ( 15 25 ) for EG, EG+SUC and control medium (GLY+SUC), respectively. The pregnant recipient animals delivered healthy calves except for five which aborted. These results indicated that EG is a suitable cryoprotectant in which to store embryos before they are directly thawed and transferred to recipients.