Impact of methanol-based composite vitrification on Oplegnathus punctatus embryo cryopreservation

Fish embryo cryopreservation is a crucial technical approach for protecting fish genetic germplasm resources. This study aimed to investigate the survival and development of Oplegnathus fasciatus embryos after equilibrium treatment with a methanol-based composite vitrification solution and subsequent cryopreservation at different low temperatures. Three methanol-based vitrification solutions-MPS (MeOH+PG+20% Suc), MDS (MeOH+DMSO+20% Suc), and MGS (MeOH+Gly+20% Suc)-were prepared at concentrations of 2, 3, and 4 mol/L. Results indicated that tail-bud stage embryos exhibited significantly higher normal development rates after equilibrium treatment compared to other developmental stages (P<0.05). Moreover, increasing the concentration of the cryoprotectants had no significant inhibitory effect on the normal development of embryos at later stages (P>0.05). Based on this, cryopreservation experiments are carried out, frozen viable embryos were obtained at 0, −20 and −40 ℃, and MPS had the best freezing effect among the three cryoprotectants. After −40 ℃ treatment, the survival rate of the 3 mol/L MPS group was the highest (46.72%±1.89%). At −196 ℃, thawed embryos maintained relatively intact structures but showed no further development during subsequent culture. Among the three cryoprotectants, embryos treated with MPS exhibited the highest structural integrity post-thawing. The cryopreservation effect of the MPS composite cryoprotectant on tail-bud stage embryos of O. fasciatus was superior to that of MDS and MGS. After cryopreservation at −40 ℃, the embryo survival rates under the three cryoprotectants at 3 mol/L concentration were higher than those at other concentrations.