Cryopreservation of mouse morulae in glycerol, sucrose and honeybee royal jelly

Abstract

Compacted mouse morulae were frozen at 0.3ºC/min. or 0.5ºC/min. from -6ºC to -24ºC or -32ºC in 10% of glycerol plus different sucrose concentrations with or without 0.1% of honeybee royal jelly. Embryos were thawed in water bath at 22ºC for 20 seconds and cryoprotectant dilution was done in three steps. Embryos were cultured in Whittens medium for 24, 48 and 72 hours at 37ºC, 5% of CO2 and 100% of humidity. The in vitro development ranged from 56.6% to 100% after 72 hours. Expanded blastocysts were transferred to pseudopregnant recipients on the third day of the estrous cycle. Viable fetuses rates for embryos frozen to -24 or -32ºC at 0.3ºC/minute in 10% glycerol + 10% sucrose, 10% glycerol + 10% sucrose + 0.1% honeybee royal jelly, 10% glycerol + 0.1% honeybee royal jelly or 10% glycerol were respectively: 28.1% and 13.6%, 48.7% and 31.9%, 28.6% and 13.2%, 20.0% and 42.4%. Viable fetuses for embryos frozen to -24ºC or -32ºC at 0.5ºC/minute in 10% glycerol + 10% sucrose or 10% glycerol + 10% sucrose + 0.1% honeybee royal jelly were respectively 29.0% and 15.3%, 48.8% and 32.0%. We can conclude that addition of 10% sucrose to 10% glycerol was efficient for embryo freezing at 0.3 or 0.5ºC/minute and plunged in liquid nitrogen at -24ºC. The honeybee royal jelly addition provided higher viable fetuses rates when embryos were cooled at 0.3 or 0.5ºC/minute and plunged in liquid nitrogen at -24ºC.