28 Effect of deuterium oxide on bovine oocyte cryotolerance

Abstract

It is known that cryopreservation triggers spindle disassembly, increased aneuploidy risk, decreased post-thaw survival, fertilization, and embryo development. We hypothesised that a treatment with D₂O before vitrification would slow down oocyte metabolism and reduce ice crystal formation by replacing water inside the cells. The aim of the study was to evaluate the effect of a 4-h treatment with different D₂O concentrations (0, 3, 15, and 30%) on cryotolerance of bovine in vitro-matured oocytes. Abattoir-derived bovine oocytes were matured in vitro for 20 h in TCM-199 medium with 15% of bovine serum (BS), 0.5 µg mL⁻¹ of FSH, 5 µg mL⁻¹ of LH, 0.8 mM l-glutamine, and 50 µg mL⁻¹ of gentamicin at 39°C with 5% of CO₂ and randomly divided into 5 experimental groups. A group of non-vitrified oocytes was used as the fresh oocyte control group, whereas the remaining oocytes were incubated for 4 h in in vitro maturation medium with 0% (vitrified control; n = 205), 3% (n = 205), 15% (n = 205), and 30% D₂O (n = 205) before vitrification. The experiment was repeated 4 times. Oocytes were denuded in HEPES-buffered TCM-199 (H199) + 5% BS and vitrified using a cryotop freezing straw. The oocytes were incubated in 200 μL of H199 + 20% BS with 7.5% ethylene glycol and 7.5% dimethyl sulfoxide for 3 min. After that, oocytes were collected in 50 μL of H199 + 20% fetal bovine serum with 15% ethylene glycol + 15% dimethyl sulfoxide and 0.5 M sucrose for 20 s and plunged into LN₂. One month later, oocytes were warmed in thawing media with decreasing concentrations of sucrose (1.35 M to 0.31 M) and then placed into in vitro maturation medium for 2 h before IVF. Matured oocytes were IVF and cultured according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-1355). Cleavage and blastocyst rates were evaluated after 7 days of culture. Data were analysed using the GLM procedure of SPSS (SPSS Inc., Chicago, IL, USA). The least statistical difference post-hoc test was used to perform statistical multiple comparison. The α-level was set at 0.05. As expected, both cleavage [60.5 ± 4.6 (fresh control); 36.9 ± 2.6 (0% D₂O); 46.3 ± 3.7 (3% D₂O); 31.6 ± 2.4 (15% D₂O); and 24.4 ± 2.6 (30% D₂O)] and blastocyst rates [25.7 ± 0.8 (fresh control); 9.0 ± 0.8 (0% D₂O); 9.0 ± 0.7 (3% D₂O); 3.6 ± 0.2 (15% D₂O); and 4.3 ± 0.8 (30% D₂O)] decreased in all vitrified groups compared with the fresh control group. Within vitrified oocytes, cleavage rate increased (P < 0.05) with 3% D₂O treatment compared with the other groups. However, pretreatment with higher (15-30%) D₂O concentrations decreased (P < 0.05) blastocyst rates of vitrified-warmed oocytes. In conclusion, a pretreatment with low concentrations (3%) of D₂O improved the cleavage rate of bovine vitrified-warmed oocytes, suggesting a potential beneficial effect, whereas deleterious effects were observed using the higher concentrations. Therefore, further studies are required to assess a potential use of D₂O to improve oocyte cryotolerance, likely testing different incubation times.