Comparison of slow- and rapid-cooling protocols for early-cleavage-stage Macaca fascicularis embryos

Cryostorage of nonhuman primate embryos by time-consuming slow-cooling methods is often limited to early cleavage stages. Effective rapid-cooling methods have been developed for many species and represent valuable tools for laboratory- and field-based studies of nonhuman primate reproductive biology. However, few rapid-cooling protocols have been applied to nonhuman primate embryos in terms of comparing various developmental stages. Here we compare slow cooling vs. two- and three-step rapid cooling of two-, four-, and eight-cell Macaca fascicularis (Mf) embryos. Rapid cooling was conducted in open pulled straws (OPS) using cooling solutions containing reduced quantities of ethylene glycol (EG) and supplemented with either of two high-molecular-weight polymers, ficoll and dextran. The survival of the slow-cooled embryos, but not the rapid-cooled embryos, was independent of embryonic stage at cryostorage. Slow cooling was associated with greater cell survival (82%) post thaw compared to warming following rapid cooling (18-29%). Slow cooling resulted in a high proportion of embryo survival (18/20; 90%) and cleavage (15/18; 83%) post thaw. Rapid cooling resulted in significantly reduced percentages of embryo survival (26-32%) and embryo cleavage in culture (29-38%) after warming. Conventional slow cooling was more effective than the rapid-cooling protocols employed in this study for cryopreservation of early-cleavage-stage Mf embryos.