Cryopreservation of fish embryos and embryonic cells

Abstract

Embryonic cells of dechorionated fish embryos dissociated in Ca*+, Mg’+ free medium were successfylly integregated to recipient embryos using a novel aggregation method for fish resulting in survival of fish chimeras. Embryonic cells were successfully cultured in sterile cell culture for up to 1 month. Experiments were carried out on the cryopreservation of pluripotent diploid embryonic cells derived from dechorionated embryos of African catfish (Clurias gariepinus) using two-step freezing and programmable freezer. Cells were equilibrated in media containing DMSO and methanol as cryoprotectants. A high percentage (84-95%) of survival has been achieved upon thawing unrelated to seeding applied. Survival was tested using fluorescein diacetate (FDA) vital staining in order to examine the membrane integrity of the cells upon thawing. In teleosts, the cryopreservation of eggs and embryos has had very limited success so far. In some cases eggs tolerated temperatures below 0°C but no one succeeded in prolonged preservation below -60°C. Several species of teleost, mostly Salmonidae were studied by other groups. We were the first to choose the African catfish as a suitable model for the examination of cryobehaviour of fish ova and embryos. In order to find the stages with the highest penetration rate we examined the permeability of the eggs towards cryoprotectants (CPAs) in different developmental stages. Following exposure of the ova to concentrated CPA solutions, the penetration of the CPAs was detected by visual observation of vitrification of the eggs plunged into liquid nitrogen in straws. Our results indicated that the permeability of unfertilized eggs is much higher than that of fertilized eggs. The permeability is sharply increased in the early morula stage and remains high later on. The vitrification is thought to be a combined effect of dehydration and penetration of CPAs. The percent of eggs vitrified following lo,30 and 60 min exposure at 23°C to 6.5 M DMSO was 4%, 87% and 100%; 6.5 M propylene glycol was O%, 90% and 100%; 6.5 M glycerol was O%, 0% and 90%; 6.0 M ethylene glycol was 0%, 0% and 83%, respectively. None of the eggs survived more than 5 min exposure to the CPA solutions studied.