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155 Oocyte Maturation in Lyophilized In Vitro Maturation Medium as a Method to Increase the Medium’s Shelf-Life

The in vitro production of bovine embryos has dramatically increased in recent years, and with it the demand of stable media with a long shelf-life. In this experiment we evaluated the impact of the freeze-dried in vitro maturation (IVM) medium (Mdry) on in vitro oocyte maturation. We compared the standard IVM and the Mdry media. Medium M199 was used as base for the IVM medium. The percentage of metaphase II oocytes and embryo production were evaluated. Media solutions (10 mL) were aliquoted into 50-mL conical tubes and lyophilized to form a powder concentrate using a Genesis freeze-dryer (VirTis, Gardener, NY, USA). Lyophilization consisted of a constant cooling from 20°C to –10°C at a constant rate of 1°C/min with a 2-h hold at –10°C before sublimation at 0°C. The Mdry medium was held at –80°C for 4 months (only serum and hormones were added before the incubation). When the IVM medium was rehydrated, the pH were adjusted to 7.4. The percentage of mature oocytes was evaluated after 24 h of maturation. The oocytes were stained with Hoechst 33342, and only oocytes with metaphase and a polar body were evaluated as matured. Abattoir-derived Holstein oocytes (n = 540) were in vitro matured (25–30/well in 400 µL) and fertilized with sexed semen, according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-1355). The oocytes were split for analysis (432 were used for IVP and 108 for maturation rate) over 6 replicates. Twenty hours after IVF, presumptive zygotes were cultured in SOF medium at 39°C with 5% CO2, 7% O2, and 88% N2. On Day 7, embryo yields were assessed. All recorded parameters were subjected to a Student’s t-test. The parameters compared were maturation rate, cleavage rate, blastocyst rate and the percentage of embryos cleaved. The α level was set at 0.05. All data were expressed as quadratic means and mean standard deviations. The results showed no differences between the 2 groups (75.9% v. 74.1%) (t = 0.37; SD = 12.69; P = 0.36; df = 5) when we compared the nuclear maturation; however, when we evaluated embryo production, we found the Mdry treatment had a higher cleavage percentage (t = 2.39; SD = 14.81; P = 0.02; df = 5) and total embryos produced (t = 2.49; SD = 5.6; P = 0.02; df = 5) compared with the control (Table 1.). These results showed that lyophilization can be a valid method to increase the shelf life of IVP media. More replicates must be done in order to understand why the freeze-dried media produced more embryos. Table 1.Mean (SD in parentheses) percentage cleavage and blastocysts