Effect of methanol-based vitrification solutions on the survival rate of cryopreserved common carp (Cyprinus carpio) embryos

Abstract

Abstract The objective of this study was to establish an optimal protocol for the vitrification of common carp embryos. Additionally, the cryoprotective efficiency of natural honey as a non-permeable cryoprotectant was also examined. Six methanol-based vitrification solutions (V1–V6) were designed to be checked for their effects on the heartbeat stage embryos using a four-step protocol. Methanol-based vitrification solutions contained 4 M methanol + one or two other permeable cryoprotectants + 20% sucrose or natural honey. Toxicity tests showed that the hatching rates of embryos after exposure to V5 and V6 were significantly higher than those of other solutions (P < 0.05). Embryos were cryopreserved in the six vitrification solutions in liquid nitrogen (LN2, -196°C) for 30 and 60 min. After thawing (in a water bath at 24°C for 30 s), all vitrification solutions produced hatched larvae. The results revealed that V6 (4 M methanol + 3 M dimethyl sulfoxide + 2 M propylene glycol + 20% honey) was the most effective for cryopreservation of common carp embryos. The highest hatching rates after storage for 30 and 60 min in LN2 were 44.76% and 17.14%, and the highest survival rates were 36.19% and 11.42%, respectively. The mean normal development rates for vitrified-thawed embryos after 30 and 60 min of storage in LN2 were 14.28% and 2.38%, respectively. As natural honey showed greater cryoprotective efficiency than sucrose for common carp embryos, it is suggested that it can be used instead of sucrose, which is traditionally used in fish embryo cryopreservation.