Effects of cryoprotectant and plunging temperature in liquid nitrogen on the in vitro and in vivo development of murine morulae

RESUMO

The effects of plunging temperature in liquid nitrogen and cryoprotectant dilution methods were evaluated for compacted mouse morulae frozen in 1.5 M ethylene-glycol (E), 1.5M propylene-glycol (P) or 1.4M glycerol (G). Morulae were equilibrated for 10 minutes in cryoprotectant solution and loaded into 0.25 ml straws with cryoprotectant solution in 3 columns (groups E1, P1, G1) or cryoprotectant in the center and PBS in the lateral columns (E2, P2). Straws were cooled at 0.5§C/min to -25 or -30§C and plunged into liquid nitrogen. Straws were thawed in water at 22§C for 20 seconds. Cryoprotectant was diluted in 3 steps for group G1 and in one step for groups E1 and P1 (direct transfer to PBS + 10 por cento FCS) and E2 and P2 (shaken to mix the 3 columns before transferring to PBS+ 10 por cento FCS). Plunging temperature had no significant effect on the proportion of morulae developed to blastocysts in vitro; this proportion was higher (p < 0.0001) in E1 (69.2 por cento) than in E2 (60.3 por cento), G1 (51.9 por cento) and combined for P1 and P2 (46.9 por cento). In second experiment, the proportion of transferred morulae that developed to viable fetuses was lower (p < 0.07) for E1-25 than for E1-30, G1-30, E2-25 or unfrozen (control) embryos (8.7, 20.0, 20.0, 17.4 and 19.8 por cento, respectively). In conclusion, the ethylene glycol diluted directly in PBS (E1) exhibit the highest rate of in vitro embryos development, but based on in vivo embryos development was more efficacious in plunging temperature at -30§C (E1-30)