title: Preliminary studies on the vitrification of red sea bream (Pagrus major) embryos.
abstract: The objective was to identify an appropriate cryoprotectant and protocol for vitrification of red sea bream (Pagrus major) embryos. The toxicity of five single-agent cryoprotectants, dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), glycerol (GLY), and methyl alcohol (MeOH), as well as nine cryoprotectant mixtures, were investigated by comparing post-thaw hatching rates. Two vitrifying protocols, a straw method and a solid surface vitrification method (copper floating over liquid nitrogen), were evaluated on the basis of post-thaw embryo morphology. Exposure to single-agent cryoprotectants (10% concentration for 15 min) was not toxic to embryos, whereas for higher concentrations (20 and 30%) and a longer duration of exposure (30 min), DMSO and PG were better tolerated than the other cryoprotectants. Among nine cryoprotectant mixtures, the combination of 20% DMSO+10% PG+10% MeOH had the lowest toxicity after exposure for 10 min or 15 min. High percentages of morphologically intact embryos, 50.6+/-16.7% (mean+/-S.D.) and 77.8+/-15.5%, were achieved by the straw vitrifying method (20.5% DMSO+15.5% acetamide+10% PG, thawing at 43 degrees C and washing in 0.5M sucrose solution for 5 min) and by the solid surface vitrification method (40% GLY, thawing at 22 degrees C and washing in 0.5M sucrose solution for 5 min). After thawing, morphological changes in the degenerated embryos included shrunken yolks and ruptured chorions. Furthermore, thawed embryos that were morphologically intact did not consistently survive incubation.
authors.affiliation: Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.
authors.forename: F H
authors.initials: FH
authors.lastname: Ding
authors.forename: Z Z
authors.initials: ZZ
authors.lastname: Xiao
authors.forename: J
authors.initials: J
authors.lastname: Li
mesh_terms: D000818:Animals; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000633:toxicity; D004338:Drug Combinations; D004625:Embryo, Nonmammalian; D008954:Models, Biological; D010865:Pilot Projects; D021541:Sea Bream / Q000196:embryology*
chemical_list: D003451:Cryoprotective Agents; D004338:Drug Combinations