Surubim-do-Paraíba oocytes viability after being exposed to different cryoprotectants

ABSTRACT: To know the non-toxic cryoprotectants to fish oocytes is of extreme importance for tests that aim to increase oocyte resistance to cold, thus allowing more advanced studies in cryopreservation. Therefore, commonly used cryoprotectants such as methanol, dimethyl sulfoxide, ethylene glycol, propylene glycol, sucrose and fructose were studied. Immature oocytes from the initial to vitelogenic (diameter <1.7 mm) and mature (diameter >1.8 mm) stages of Steindachneridion parahybae were evaluated. Four distinct experiments were performed, three using immature oocytes and one using oocytes at the mature stage. For each oocyte stage, the best maintenance solution to be used: Hank or 50% L15 and; viability after baths for 30min (room temperature) at cryoprotectant concentrations ranging from 0.25 to 4M were evaluated. Different tests were used to evaluate oocyte viability: in vitro maturation followed by observation of germinal vesicle breakdown (only for immature oocytes), Trypan Blue staining (all stages) and fertilization and hatching rates (mature stage only). Results showed that the toxic effect of cryoprotectants on oocytes generally increases with increasing concentrations. Sensitivity of oocytes to cryoprotectants increases according to the stage of development, with mature oocytes being more sensitive. Sucrose, fructose, methanol, propylene glycol and dimethyl sulfoxide can be used as cryoprotectants for S. parahybae oocytes.

摘要：明确对鱼类卵母细胞无毒性的低温保护剂，对于提升卵母细胞抗寒能力的相关实验至关重要，同时可为鱼类卵母细胞冷冻保存领域的深入研究奠定基础。为此，本研究选取甲醇、二甲基亚砜、乙二醇、丙二醇、蔗糖以及果糖等常用低温保护剂开展实验。本研究对帕拉伊巴似舒氏鲶（Steindachneridion parahybae）处于初始发育至卵黄发生期（直径＜1.7 mm）以及成熟期（直径＞1.8 mm）的未成熟卵母细胞进行了评估。本研究共开展4组独立实验：3组以未成熟卵母细胞为实验材料，剩余1组使用成熟卵母细胞。针对不同发育阶段的卵母细胞，本研究分别评估了其适配的最优维持液（汉克液（Hank）或50% L15培养基），以及在浓度范围为0.25~4 M的低温保护剂中室温浸泡30 min后的细胞活力。本研究采用多种检测方法评估卵母细胞活力：对于未成熟卵母细胞，采用体外成熟培养后观察生发泡破裂（germinal vesicle breakdown, GVBD）情况的方法；对于所有发育阶段的卵母细胞，均采用台盼蓝染色（Trypan Blue staining）法；仅针对成熟卵母细胞，则检测其受精率与孵化率。实验结果表明，低温保护剂对卵母细胞的毒性作用通常随浓度升高而增强。卵母细胞对低温保护剂的敏感性随发育阶段推进而升高，其中成熟卵母细胞的敏感性最高。蔗糖、果糖、甲醇、丙二醇以及二甲基亚砜可作为帕拉伊巴似舒氏鲶（S. parahybae）卵母细胞的低温保护剂。