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