A study of sucrose for removal of cryoprotectant from frozen bovine embryos

Abstract

Cryoprotectant removal within the straw, using sucrose, has previously been reported. Experiment A using dimethyl sulfoxide (DMSO) and Experiment B using glycerol were designed to compare three concentrations of sucrose and cryoprotectant for embryo survival. A 3 x 3 experimental design was employed using 3 levels of cryoprotectant (0.5, 1.0 and 1.5 M) and sucrose (0.25, 0.5 and 1.0 M). A tenth treatment (6-step dilution) served as a control. Grade 1 and 2 embryos were thawed in 0.5 ml straws in a 37(DEGREES)C water bath after 1 min in air at 20(DEGREES)C. Post thaw viability was assessed using fluorescein diacetate and cultures of embryos in Ham's F-10 medium under paraffin oil at 37(DEGREES)C in an atmosphere of 5% CO(,2) in air. In experiments A and B: 25/187 (13.4%) and 33/194 (17.0%) frozen and thawed embryos developed at least 1 morphological stage, 12/187 (6.4%) and 11/194 (4.7%) held or maintained grade and/or stage of development (GREATERTHEQ) 48 hrs, while 3/187 (1.6%) and 2/194 (1%) hatched in vitro. Due to similarly low survival in all treatments there was no significant correlation between treatment and embryo viability. Optimal cryoprotectant and sucrose concentrations could not be determined, however, the main effect means and least squares means for cryoprotectants and sucrose, indicate the medium concentration of cryoprotectant (1.0 M) and the lower concentration of sucrose (0.25 M) favor development in vitro after thawing. Post rehydration quality grades and fluorescein diacetate (FDA) scores suggest that many embryos survived freezing and thawing in experiments A and B; however, few developed in culture. Following preparation for freezing in 1.5 M glycerol using modified phosphate buffered saline (MPBS) and 20% hyclone newborn calf serum (NBCS), forty-eight grade 1, 2 and 3 embryos were equilibrated 40 min and rehydrated using (a) 0.7 M sucrose, (b) 1.15 M sucrose or (c) 6-step dilution, 10 min each. Although poor quality embryos were included in this study, 13/16 (81%) and 12/16 (75%) hatched during culture in treatments a-c, respectively. These data suggest that bovine embryos survive removal of cryoprotectant with sucrose, but leave thawing rate as suspect in causing low in vitro survival of frozen/thawed embryos in straws.