Abstract
The aim of the present study was to compare the efficiency of vitrification and slow freezing techniques for the cryopreservation of zebrafish ovarian tissue containing immature follicles. In Experiment 1, assessment of cell membrane integrity by trypan blue exclusion staining was used to select the best cryoprotectant solution for each cryopreservation method. Primary growth (PG) oocytes showed the best percentage of membrane integrity (63.5 ± 2.99%) when SF4 solution (2 M methanol + 0.1 M trehalose + 10% egg yolk solution) was employed. The vitrification solution, which presented the highest membrane integrity (V2; 1.5 M methanol + 5.5 M Me2SO + 0.5 M sucrose + 10% egg yolk solution) was selected for Experiment 2. Experiment 2 aimed to compare the vitrification and slow freezing techniques in the following parameters: morphology, oxidative stress, mitochondrial activity, and DNA damage. Frozen ovarian tissue showed higher ROS levels and lower mitochondrial activity than vitrified ovarian tissue. Ultrastructural observations of frozen PG oocytes showed rupture of the plasma membrane, loss of intracellular contents and a large number of damaged mitochondria, while vitrified PG oocytes had intact mitochondria and cell plasma membranes. We conclude that vitrification may be more effective than slow freezing for the cryopreservation of zebrafish ovarian tissue.
Highlights
The most widely used fish model is zebrafish (Danio rerio), which presents 12,719 genes in common with humans[1], and shows high fertility[2] and embryonic transparency[3]
The membrane integrity of Primary growth (PG) oocytes in the control group was significantly higher compared to all vitrification solutions; there was no significant difference among the treatments (Fig. 1)
When ovarian tissue fragments were frozen in SF1 and SF3, the membrane integrity decreased to 41.5 ± 21.0% and 43.75 (P < 0.05),respectively
Summary
The most widely used fish model is zebrafish (Danio rerio), which presents 12,719 genes in common with humans[1], and shows high fertility[2] and embryonic transparency[3]. We obtained a high viability of immature follicles (76% in stages I and 43% in stages II) following zebrafish ovarian tissue vitrification[12]. The two most commonly used cryopreservation methods for the preservation of animal germplasm are slow freezing and vitrification Both methods are based on the same principles of protecting cells from damage due to intracellular ice formation, excessive cell dehydration and changes in solute concentrations. No study has compared vitrification and slow freezing techniques in the cryopreservation of zebrafish ovarian tissue fragments. The major aim of the present study was to compare the efficiency of vitrification and slow freezing techniques in the cryopreservation of zebrafish ovarian tissue containing immature follicles
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