Marques Lis S, Fossati Ana A, Leal Martinha S, Rodrigues Rômulo B, Bombardelli Robie A, Streit Danilo P
Universidade Federal do Rio Grande do Sul (UFRGS), Programa de Pós-Graduação em Zootecnia, 91540-000, Porto Alegre, RS, Brazil.
Universidade Federal do Rio Grande do Sul (UFRGS), Programa de Pós-Graduação em Zootecnia, 91540-000, Porto Alegre, RS, Brazil.
Cryobiology. 2018 Jun;82:118-123. doi: 10.1016/j.cryobiol.2018.03.009. Epub 2018 Mar 26.
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 MeSO + 2.5 M etilenoglycol +0.5 M sucrose (VS3; 66.43 ± 4.68%) and 1.5 M methanol + 5.5 M MeSO + 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 (r = 0.78) and membrane (r = 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.
对含有未成熟卵母细胞的卵巢组织进行玻璃化冷冻，为保护濒危物种和水生物种的遗传多样性提供了一种重要手段。因此，本研究的主要目的是通过组织学分析、两种染色方法（台盼蓝或双醋酸荧光素联合碘化丙啶）以及线粒体活性检测（MTT检测）来评估卵巢组织玻璃化冷冻后初级生长（PG）卵母细胞的活力。此外，还进行了卵母细胞组织形态测量，以评估玻璃化冷冻后的形态学参数以及与PG卵母细胞损伤（细胞核和/或细胞膜）发生情况的关系。使用台盼蓝染色或FDA + IP染色对玻璃化冷冻的卵母细胞进行评估时，未发现显著差异。通过组织学分析评估的卵母细胞活力显示，玻璃化溶液2.0 M MeSO + 2.5 M乙二醇 + 0.5 M蔗糖（VS3；66.43 ± 4.68%）和1.5 M甲醇 + 5.5 M MeSO + 0.5 M蔗糖（VS5；74.14 ± 3.71%）的活力率最低。在MTT检测中也观察到了类似结果，与VS1（2.39 ± 0.14）、VS2（1.78 ± 0.06）和VS4（2.34 ± 0.19）相比，VS3（1.63 ± 0.12）和VS5（1.58 ± 0.09）的平均值最低（P = 0.0002）。在通过组织学评估细胞膜损伤时，玻璃化冷冻的卵母细胞与对照组之间没有差异。然而，在VS3（26.00 ± 5.55）和VS5（26.00 ± 5.55）处理组中观察到细胞核损伤的比例最高。玻璃化冷冻后卵母细胞直径没有变化；然而，对照组的细胞核直径显著更高（49.03 ± 1.07）。通过组织学分析得出的卵母细胞活力与玻璃化冷冻/复温后细胞核（r = 0.78）和细胞膜（r = 0.45）损伤的发生呈正相关。对南美脂鲤卵巢组织进行玻璃化冷冻后获得的PG卵母细胞具有较高的活力，这表明本文所采用的方案可能成功应用于其他硬骨鱼物种以用于食物生产。
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