Hydrogel encapsulation as a handling and vitrification tool for zebrafish ovarian tissue.

Abstract

Zebrafish is an important animal model, thousands lines have been developed, thus having a great need for their preservation. However, the cryopreservation of fish oocytes is still limited and needs improvement. The sodium alginate hydrogel, in addition to providing support for the cells, has been shown to be a potential cryoprotectant. Therefore, the aim of this study was to evaluate the sodium alginate hydrogel encapsulation technique efficiency during zebrafish ovarian tissue vitrification. The encapsulation methodology was standardized in the first experiment. In Experiment 2, we evaluated four vitrified groups: standard protocol without encapsulation (VS); encapsulated with cryoprotectants (VS1-A); encapsulated with half the cryoprotectants concentration (VS2-A); encapsulated without cryoprotectants (VA). VS treatment (54.6 ± 12.3%; 23.7 ± 9.9%; 12.6 ± 5.0%) did not differ from the VS1-A and VA showed a lower membrane integrity percentage (1.2 ± 1.4%; 0.3 ± 0.6%; 0.5 ± 1.5%). Mitochondrial activity was significantly greater in non-encapsulated treatment (VS) when compared to the encapsulated treatments. VS1-A and VS obtained the lowest lipid peroxidation (39.4 ± 4.4 and 40.5 ± 3.3 nmol MDA/mg respectively) in which VS was not significantly different from the VS2-A treatment (63.6 ± 3.1 nmol MDA/mg), unlike, VA obtained the highest lipid peroxidation level (124.7 ± 7.9 nmol MDA/mg). The results obtained in this study demonstrate that the sodium alginate hydrogel encapsulation technique did not have a cryoprotective action, but maintained the membrane integrity when used the standard concentration of cryoprotectants. However, halving the cryoprotectant concentration of fragments encapsulated in alginate hydrogel did not cause an increase in lipid peroxidation. In addition, it provided support and prevented the oocytes from loosening from the tissue during the vitrification process, being an interesting alternative for later in vitro maturation.