Effects of cryopreservation at various temperatures on the survival of kelp grouper (Epinephelus moara) embryos from fertilization with cryopreserved sperm.

Fish embryo cryopreservation is highly important for the long-term preservation of genomic and genetic information; however, few successful cases of fish embryo cryopreservation have been reported over the past 60 years. This is the first study to use Epinephelus moara embryos from fertilization with cryopreserved sperm as experimental material. Embryos that developed to the 16-22 somite stage and tail-bud stage were treated with the vitrification solution PMG3T according to a five-step equilibration method and cryopreserved at various temperatures and storage duration. Only 19.9 ± 9.2% of 16-22 somite stage embryos and 1.3 ± 1.1% of tail-bud stage embryos survived when cooled at 4 °C for 60 min. In total, 8.0 ± 3.0% of 16-22 somite stage embryos survived when cooled at -25.7 °C for 30 min, 22.4 ± 4.7% of tail-bud stage embryos survived after 45 min of cooling at -25.7 °C, and none survived after 60 min. Only 2.0 ± 2.7% of embryos survived when cryopreserved at -140 °C for 20 min. However, 9.7% of tail-bud stage embryos survived after cryopreservation in liquid nitrogen (-196 °C) for 2 h. Most surviving embryos developed normally. Embryonic volume decreased and spherical segments appeared when embryos were treated with higher concentrations of vitrification solution. Additionally, the volume recovered gradually after rinsing with sucrose and seawater. This is the first estimate of the survival of E. moara embryos and larvae after cryopreservation. These findings provide a foundation for further explorations of fish embryo cryopreservation techniques.

Effects of cryopreservation on the survival rate of the seven-band grouper (Epinephelus septemfasciatus) embryos.

The effects of cryopreservation and the vitrification solution on the embryo hatchability of the seven-band grouper Epinephelus septemfasciatus were evaluated in this study. Six small molecule cryoprotectants (PG, MeOH, Gly, DMF, DMSO and EG) and four macromolecular cryoprotectants (glucose, fructose, sucrose and trehalose) were used to determine the embryo toxicity levels. Results showed that the embryo survival rate was higher when the PM (24% PG + 16% MeOH):Gly ratios were 3:1 and 4:1. Further experiments showed that the embryo survival rates in PMG3S (35% PMG3 + 5% sucrose) and PMG3T (35% PMG3 + 5% trehalose) were relatively higher, which are 29.24 ± 10.81% and 27.01 ± 3.39%, respectively. When treated with PMG3S and PMG3T by using 5-step method, embryos at somite stage and tail-bud stage shrank in the first 6 min and gradually recovered in volume to the original. This indicated the successful permeation of the vitrification solutions into cells. Then, embryos at the embryoid body formation stage, the somite stage and the tail-bud stage were cryopreserved with PMG3S and PMG3T. In total, 82 floating embryos were obtained, 14 of which developed further, with 8 embryos at the tail-bud stage developing to the heartbeat stage, 4 embryos at the body formation stage development to the somite stage, and 2 embryos at the somite stage hatched to larval fish.