94 Embryonic development and cryogenic viability of bovine IVP blastocysts under the impact of Mito-TEMPO
M. Schreiber A , J. Kurzella B , D. Salilew-Wondim A , D. Teuteberg A , C. Blaschka A and M. Hoelker AA
B
In vitro-produced bovine embryos still exhibit inferior rates of development, pregnancy, and cryogenic viability in comparison to in vivo counterparts. It has already been shown that the addition of mitochondrial active antioxidants like Mito-TEMPO (Sigma) to the media exerts a positive effect on developmental rates and cryogenic fitness of porcine embryos. The current study’s goal was to ascertain if adding the antioxidant Mito-TEMPO to the maturation or to the culture medium may have an impact on the early embryonic development and cryogenic viability of bovine blastocysts. For the study, cumulus–oocyte complexes (COCs) were obtained from the ovaries of slaughtered cows by slicing method. In the first experimental run, maturation was performed for 22 h in 4-well plates (NUNC, 400 µL, 39°C, 5% CO2, 20% O2), with 50–70 COCs each randomly assigned to a control group (TCM + Suigonan) and a treatment group (TCM + Suigonan +1 µM Mito-TEMPO). In contrast, in the second experimental run, the maturation was performed exclusively with TCM + Suigonan under the above parameters. For the IVF frozen semen was purified (SpermFilters®, IVF Bioscience) and added to the matured oocytes at a concentration of 2 × 106 cells/mL (Fert.-TALP.-medium, 400 µL, NUNC). Nineteen hours after the start of IVF, the COCs of the first experimental run (n = 2475) were denuded by vortexing and subsequently cultured until Day 8 of development (synthetic oviductal fluid + 0.3% BSA, 4-well dish, 400 µL, oil overlay, 39°C, 5% CO2, 20% O2). In contrast, in the second experimental run, the fertilized oocytes (n = 3150) were cultured in a control group (SOFaa + 0.3% BSA) and in a treatment group (SOFaa 0.3% BSA + 1 µM Mito-TEMPO). Day 7 blastocysts of both experimental groups were individually vitrified using BO-VitriCool™-media (IVF Bioscience) and the Cryotop®-vitrification system (Kitazato). The warming (BO-VitriWarm™-media, IVF Bioscience) of the vitrified blastocysts was followed by a postwarming culture for 72 h to determine viability, expansion and hatching rates. The results of the first experimental run, where the maturation medium was supplemented with 1 µM Mito-TEMPO, showed no significant effect (ANOVA, P > 0.05) of Mito-TEMPO on both division and blastocyst rates. In contrast, the blastocysts derived from oocytes maturated with Mito-TEMPO showed a significantly (P < 0.05) higher hatching rate (ø 21%) after vitrification than the control group (10.9%). In the second experimental run, in which the culture medium was supplemented with 1 µM Mito-TEMPO, no significant effect of Mito-TEMPO was detected in the cleavage and blastocysts rate. In contrast, in the postwarming culture, the blastocysts cultured with Mito-TEMPO were shown to have a statistically significant (P < 0.05) higher expansion rate (control: 66%, treatment: 75%) and a higher hatching rate (control: 9.5%, treatment: 20%). The results of both experimental runs thus confirm our hypothesis that the antioxidant Mito-TEMPO exerts a positive influence on embryonic development and the survival rates after vitrification.
