Cryopreservation of Mammalian Embryos

Abstract

Early investigations of the effects of cooling and rewarming cells employed both spermatozoa and ova as experimental material. Spermatozoa were selected because of the ease of collection, inherent motility, and small size. Oocytes were employed mainly for their large size which allowed direct morphological observations. In 1776, Spallanzani investigated the effects of “winter snow and cold” on stallion semen and silkworm eggs. Upon warming, the spermatozoa were “reactivated” and became motile. This was the first report of the successful cold storage of sperm cells. It was not until 1938 that Jahnel, working on the problem of syphilis, found that sperm cells cooled to -79°C retained some motility after 40 days of storage. Luyet and Hodapp (1938) used the vitrification technique of Stiles (1930) to preserve frog spermatozoa successfully. In 1940, Shettles found that survival rates varied among semen samples; however, no greater that 10% viability was maintained in any sample. Hoagland and Pincus (1942) extended Shettles’ findings that some males did not produce semen samples suitable for cryopreservation and applied vitrification to human semen with favorable results. Parkes (1945) reported that spermatozoa survived at higher rates when large compared to small volumes were cooled. Parkes disregarded the physical law that cooling rate is proportional to surface area to volume ratio, but suggested some mechanism involving the semen surface area exposed to air as being critical to survival rate. Finally, Polge et al. (1949) discovered the cryoprotectant nature of glycerol. This discovery led to semen storage in farm animals and in 1953 to the first attempt to store human sperm (Sherman, 1973).