Cryopreservation of donkey embryos by the cryotop method: Effect of developmental stage, embryo quality, diameter and age of embryos

Abstract

Cryopreservation of embryos has the potential to become a valuable tool for the conservation of endangered donkey breeds. However, there are several factors that can affect cryosurvival of embryos. This study evaluates the effectiveness of the Cryotop method to vitrify donkey embryos and factors affecting the survival of vitrified-warmed embryos. Day 6–8 embryos were measured and morphologically evaluated. Embryos were then vitrified-warmed using the Cryotop technique. After 24 h post-warming, the embryos were measured and evaluated for their morphology, development and viability (Propidium Iodide-Hoechst 33342 dyes). A total of 25 embryos were used, of which 17 were classified as Grade 1 (excellent), 5 as Grade 2 (good) and 3 as Grade 3 (fair). Based on their diameter, embryos were grouped as follows: ≤220 μm (n = 10), >220–300 μm (n = 8), and >300 μm (n = 7). Post-warming survival of vitrified embryos was similar (P > 0.05) to the control fresh embryos, regardless of embryonic diameter, developmental stage, and age of the embryos before vitrification. However, the proportion of embryos that survived vitrification procedures was numerically higher but not significantly different (P > 0.05) for Day 7 embryos (84.6%). The ability of Grade 1 (70.6%) and 2 (80%) embryos to survive vitrification procedures was higher (P = 0.0214) than those of Grade 3 (0%). The proportion of dead cells in Grade 3 embryos (56.5%) was higher (P < 0.01) than that of Grade 1 (3.2%) and 2 (1.5%) embryos. In conclusion, the Cryotop technique seems to be useful for Grade 1 and 2 donkey embryos. It is likely that donkey embryos show similar survival rates after vitrification in Cryotops irrespective of age, diameter and development stage.