Oocytes are highly sensitive to cryopreservation, which frequently results in an irreversible loss of developmental competence. We examined the effect of membrane-permeable trehalose on the freezing ability of feline oocytes matured in vitro. In Experiment 1, intracellular trehalose (trehalose hexaacetate; Tre-(OAc)) was synthesized from trehalose precursor and subjected to spectroscopic characterization. The membrane permeability of the Tre-(OAc) was investigated by incubating oocytes with different concentrations of Tre-(OAc) (3, 15, and 30 mM). Optimum concentration and the toxicity of Tre-(OAc) were assessed in Experiment 2. The effects of Tre-(OAc) on freezing ability in terms of apoptotic gene expression and developmental competence of in-vitro matured oocytes were examined in Experiments 3 and 4, respectively. The Tre-(OAc) permeated into the ooplasm of cat oocytes in a dose- and time-dependent manner. The highest concentration of intracellular trehalose was detected when the oocytes were incubated for 24 h with 30 mM Tre-(OAc). For the toxicity test, incubation of oocytes with 3 mM Tre-(OAc) for 24 h did not affect maturation rate and embryo development. However, high doses of Tre-(OAc) (15 and 30 mM) significantly reduced maturation and fertilization rates (p < 0.05). In addition, frozen-thawed oocytes treated with 3 mM Tre-(OAc) significantly upregulated anti-apoptotic (BCL-2) gene expression compared with the control (0 mM) and other Tre-(OAc) concentrations (15 and 30 mM). Oocyte maturation in the presence of 3 mM Tre-(OAc) prior to cryopreservation significantly improved oocyte developmental competence in terms of cleavage and blastocyst rates when compared with the control group (p < 0.05). Our results lead us to infer that increasing the levels of intracellular trehalose by Tre-(OAc) during oocyte maturation improves the freezing ability of feline oocytes, albeit at specific concentrations.
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