123 In Vitro Embryo Production in Lyophilized In Vitro Culture Medium as a Method to Increase the Medium's Shelf-Life

Abstract

The worldwide production of livestock embryos requires stable medium with long shelf life. In this experiment, we evaluated the impact of the freeze-dried in vitro culture (IVC) medium (Mdry) on in vitro embryo production. We compared the standard IVC and Mdry media for cleavage rate and embryo production. 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. Mdry medium were held at –80°C for 4 months. When the IVC medium was rehydrated, the pH were adjusted to 7.4. Abattoir-derived Holstein oocytes (n = 618, in 7 replicates) were in vitro matured and fertilized with sexed semen, according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-1355). Twenty hours after IVF, presumptive zygotes were cultured in SOF medium with 5% BS 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 Chi-Square Test 2 × 2. The parameters compared were percent cleavage, blastocysts, and embryos/cleaved. The α level was set at 0.05. All data were expressed as quadratic means and mean standard errors. The results (Table 1) showed not a statistical difference between control and Mdry. The Mdry had a higher percentage of cleaved zygotes (65.4% v. 53.4%) but not enough for a statistical difference. However, when we compared embryo production, there was no difference between treatments. The ratio between blastocysts and cleaved embryos was higher in the control group but not significant according to our selected α level. These results indicate that it is possible lyophilize IVC medium without interfering with the potential quality of the medium. Further studies will be needed to better understand the positive effect of the lyophilization on the cleavage rate. Table 1.Mean (SD in parentheses) percentage cleavage and blastocysts