海藻糖对牛未成熟卵母细胞玻璃化冷冻的影响

doi:10.16431/j.cnki.1671-7236.2022.01.024

摘要/Abstract

摘要： [目的] 研究胞外冷冻保护剂海藻糖对玻璃化冷冻-解冻后牛未成熟卵母细胞形态、活性线粒体分布和核成熟的影响。[方法] 将未成熟卵母细胞随机分为新鲜组、不同浓度海藻糖（0.25、0.5和1 mol/L）组和0.5 mol/L蔗糖组共5组。新鲜组作为玻璃化冷冻的对照组，海藻糖和蔗糖组均采用两步法进行玻璃化冷冻和解冻处理。在冷冻的第1步不添加海藻糖和蔗糖，在冷冻的第2步和解冻的第1步分别添加对应浓度的海藻糖和蔗糖，在解冻的第2步海藻糖和蔗糖浓度均减半。解冻后，统计卵母细胞形态正常率；用JC-1染色检测卵母细胞的活性线粒体分布区域和荧光强度；体外成熟培养24 h后，统计卵母细胞的核成熟率。[结果] 玻璃化冷冻组卵母细胞的形态正常率和核成熟率均显著低于新鲜组卵母细胞（P<0.05），0.5 mol/L海藻糖组卵母细胞形态正常率和核成熟率均显著高于0.5 mol/L蔗糖组及0.25和1 mol/L海藻糖组（P<0.05）；0.5 mol/L海藻糖组卵母细胞的活性线粒体均匀分布在细胞膜内侧区域，在绿光和蓝光下分别呈现强的红色荧光和黄绿色荧光，且荧光强度高于其他玻璃化冷冻组卵母细胞，但仍低于新鲜组卵母细胞；0.25 mol/L海藻糖组卵母细胞的活性线粒体分布区域少于、荧光强度低于0.5 mol/L海藻糖组，蓝光下其荧光为绿色并呈现不连续的散点分布，0.5 mol/L蔗糖组和1 mol/L海藻糖组卵母细胞活性线粒体分布区域极少，蓝光下其荧光为暗绿色。[结论] 0.5 mol/L海藻糖是进行牛未成熟卵母细胞玻璃化冷冻的最佳浓度。

关键词: 牛未成熟卵母细胞; 海藻糖; 玻璃化冷冻; 活性线粒体; 核成熟

Abstract: [Objective] The study was aimed to evaluate the effect of trehalose as extracellular cryoprotectant on the morphology, active mitochondrial distribution and nuclear maturation of vitrified-thawed bovine immature oocytes. [Method] The immature oocytes were randomly divided into fresh group, different concentrations (0.25, 0.5 and 1 mol/L) of trehalose and 0.5 mol/L sucrose groups. The fresh group was used as the control group of vitrification, and the trehalose and sucrose groups were vitrified and thawed by two steps. Trehalose and sucrose were not added in the first step of freezing, trehalose and sucrose of corresponding concentration were added in the second step of freezing and the first step of thawing respectively, and trehalose and sucrose concentrations were halved in the second step of thawing. After warming, the normal rate of oocyte morphology was counted, oocytes from trehalose, the distribution area and fluorescence intensity of active mitochondria in oocytes were detected by JC-1 staining, and the nuclear maturation rate of oocytes was counted after 24 h of in vitro maturation culture. [Result] The morphological normal and nuclear maturation rate of oocytes in vitrification groups were significantly lower than those in fresh group (P<0.05), the percentage of morphological normal and nuclear maturation rate of oocytes vitrified in the presence of 0.5 mol/L trehalose were significantly higher than those of 0.5 mol/L sucrose, 0.25 and 1 mol/L trehalose (P<0.05). The active mitochondria of oocytes in 0.5 mol/L trehalose group were evenly distributed in the inner region of cell membrane, which showed strong red fluorescence and yellow green fluorescence under green and blue light, respectively. The fluorescence intensity in 0.5 mol/L trehalose group was higher than that of other vitrified oocytes, but still lower than that of fresh oocytes. The distribution area and fluorescence intensity of active mitochondria in 0.25 mol/L trehalose group were lower than those in 0.5 mol/L trehalose group, and its fluorescence was green and showed discontinuous scattered distribution under blue light. The distribution area of oocyte active mitochondria in 0.5 mol/L sucrose group and 1 mol/L trehalose group was very few, and its fluorescence was dark green under blue light. [Conlusion] 0.5 mol/L was considered the optimum concentration of trehalose to be used in solutions prepared for vitrification of bovine immature oocytes.

Key words: bovine immature oocyte; trehalose; vitrification; active mitochondrial; nuclear maturation

中图分类号:

S823

李晓霞, 肖红卫, 曹平华, 徐志谦, 张芳, 张志阳, 荆鹏华, 禹学礼, 栗颖华. 海藻糖对牛未成熟卵母细胞玻璃化冷冻的影响[J]. 中国畜牧兽医, 2022, 49(1): 224-231.

LI Xiaoxia, XIAO Hongwei, CAO Pinghua, XU Zhiqian, ZHANG Fang, ZHANG Zhiyang, JING Penghua, YU Xueli, LI Yinghua. Effects of Trehalose on Vitrification of Bovine Immature Oocytes[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 224-231.

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