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.
Subject(s)
Cryopreservation , Freezing , Ovary/physiology , Vitrification , Zebrafish/physiology , Animals , Antioxidants/metabolism , Cell Membrane/drug effects , Cryoprotective Agents/pharmacology , DNA Damage , Female , Mitochondria/drug effects , Mitochondria/metabolism , Oocytes/cytology , Oocytes/drug effects , Oocytes/ultrastructure , Ovary/drug effects , Ovary/ultrastructure , Reactive Oxygen Species/metabolismABSTRACT
Vitrification of ovarian tissue containing immature oocytes provides an important tool for protecting the endangered species and genetic diversity in aquatic species. Therefore, the main objective was to assess primary growth (PG) oocytes viability following ovarian tissue vitrification using histological analysis, two staining protocols (trypan blue or fluorescein diacetate combined with propidium iodide) and mitochondrial activity assay (MTT assay). In addition, oocyte histomorphometry was performed to evaluate the morphometric parameters after vitrification and the relationship with the occurrence of damage (nucleus and/or membrane) in PG oocytes. There was no significant difference among the vitrified oocytes using trypan blue dye or FDA + IP staining. Oocyte viability assessed using histological analysis showed that vitrification solution 2.0 M Me2SO + 2.5 M etilenoglycol +0.5 M sucrose (VS3; 66.43 ± 4.68%) and 1.5 M methanol + 5.5 M Me2SO + 0.5 M sucrose (VS5; 74.14 ± 3.71%) had the lowest viability rate. Similar results were observed in MTT assay where VS3 (1.63 ± 0.12) and VS5 (1.58 ± 0.09) had the lowest averages when compare with VS1 (2.39 ± 0.14), VS2 (1.78 ± 0.06) and VS4 (2.34 ± 0.19) (P = 0.0002). In membrane damage evaluation by histology, there was no difference among vitrified oocytes and control. However, the highest percentages of nucleus damage were observed in treatments VS3 (26.00 ± 5.55) and VS5 (26.00 ± 5.55). Oocyte diameter did not change after vitrification; however, nucleus diameter was significantly higher in control group (49.03 ± 1.07). Oocyte viability by histological analysis was positive-correlated to the occurrence of nucleus (r2â¯=â¯0.78) and membrane (r2â¯=â¯0.45) damage after vitrification/warming. The high viability of PG oocytes obtained after ovarian tissue vitrification of Piaractus mesopotamicus suggests that the protocol applied here might be used successfully in other teleost species for food production.
