Effects of supplemented intra- and extra-cellular trehalose on cryopreservation of feline oocytes

Abstract

Study 1 aimed at examining the effects of different stages of oocyte maturation and trehalose concentrations on freezing ability and developmental capability of feline oocytes. The immature and matured feline oocytes were cryopreserved and thawed. They were assessed for developmental competence. The effects of different concentrations of trehalose (0, 0.125, 0.25, 0.5, 1 M) on the freezing ability of feline oocytes were tested. The cryopreservation generally induced poor embryo development, while immature oocytes were more susceptible to cryodamage compared with the matured oocytes (P<0.05). Incubation of oocytes with high concentrations of trehalose (0.5, 1 M) significantly reduced the developmental competence. Optimal concentrations of trehalose during cryopreservation of immature and matured oocytes were different (0.125 M and 0.25 M, respectively). It is concluded that stages of meiotic maturation affected the freezing ability of feline oocytes. Optimal concentration of trehalose was differently required for immature and matured feline oocytes.

Study 2 aimed at examining the effect of membrane-permeable trehalose on the freezing ability of feline oocytes matured in vitro. intracellular trehalose (trehalose hexaacetate; Tre-(OAc)6) was synthesized from trehalose precursor and subjected to spectroscopic characterization. The membrane permeability of the Tre-(OAc)6 was investigated by incubating oocytes with different concentrations of Tre-(OAc)6 (3, 15, and 30 mM) and optimum concentration and the toxicity of Tre-(OAc)6 were subsequently assessed. The effects of Tre-(OAc)6 on freezing ability in terms of apoptotic gene expression and developmental competence of in-vitro matured oocytes were examined, respectively. The Tre-(OAc)6 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)6. For the toxicity test, incubation of oocytes with 3 mM Tre-(OAc)6 for 24 h did not affect maturation rate and embryo development. However, high doses of Tre-(OAc)6 (15 and 30 mM) significantly reduced maturation and fertilization rates (P < 0.05). In addition, frozen-thawed oocytes treated with 3 mM Tre-(OAc)6 significantly upregulated anti-apoptotic (BCL-2) gene expression compared with the control (0 mM) and other Tre-(OAc)6 concentrations (15 and 30 mM). Oocyte maturation in the presence of 3 mM Tre-(OAc)6 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)6 during oocyte maturation improves the freezing ability of feline oocytes, albeit at specific concentrations.

Study 3 aimed at examining the effects of combination of intra-cellular (Tre-(OAc)6) and extra-cellular (α,α-trehalose) trehalose on freezing ability of feline oocytes. Optimal concentrations of Tre-(OAc)6 (0, 1.5, and 3 mM) and trehalose (0, 0.125, 0.25, and 0.5 M) were investigated for cryopreservation of matured feline oocytes. The combination of the two optimal concentrations were subsequently examined. The results showed that 3 mM of Tre-(OAc)6 improved post-thawed developmental competence, while extra-cellular trehalose at 0.25 M significant enhanced freezing ability of feline oocytes (P<0.05). The combination of intra- and extra-cellular trehalose significantly improved developmental capability of frozen-thawed matured feline oocytes compared to non-trehalose control.