101 Effect of Mito-TEMPO on embryonic development and cryogenic viability of bovine in vitro-derived blastocysts
M. Schreiber A , J. Kurzella B , D. Salilew-Wondim B , D. Teuteberg A , M. Hoelker A and C. Blaschka AA Department of Animal Science, Biotechnology & Reproduction in Farm Animals, University of Goettingen, Goettingen, Germany
B Institute of Animal Science, Animal Breeding, University of Bonn, Bonn, Germany
Reproduction, Fertility and Development 35(2) 177-177 https://doi.org/10.1071/RDv35n2Ab101
Published: 5 December 2022
© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
Despite many efforts, bovine embryos produced in vitro are still characterised by lower development rates, lower pregnancy rates, and reduced cryogenic viability compared to their ex vivo-derived counterparts. With respect to cryogenic viability, it has been shown recently that the addition of antioxidants to the medium exerts positive effects on reactive oxygen species (ROS) levels, developmental rates, and cryogenic fitness of bovine in vitro-derived embryos. Therefore, we speculate that a mitochondrial active antioxidant such as Mito-TEMPO (Sigma, SML0737) affects these parameters as well. Consequently, the present study aimed to investigate the effect of Mito-TEMPO supplementation in the culture medium on early embryonic development and cryogenic viability of subsequent in vitro-generated bovine blastocysts. A total of nine replicates were performed for this study. Cumulus-oocyte complexes (COC) were obtained by aspiration from ovaries collected at a local slaughterhouse. Subsequent maturation was performed in groups of 50–70 COCs in TCM199-medium for 22 h in four-well plates (400 µL culture volume without oil overlay, 39°C, 5% CO2, 20% O2). In vitro fertilisation was conducted using frozen-thawed and purified semen added to the matured oocytes at a concentration of 2 × 106/mL. Following fertilisation, presumptive zygotes were cultured for up to eight days in SOFaa + 0.3% bovine serum albumin medium. While half of the presumptive zygotes were allocated to culture medium supplemented with additional Mito-TEMPO (1 µM) the remaining ones cultured without additional Mito-TEMPO served as CONTROL. On Day 7 of culture, quantification of intracellular ROS levels was performed in 23 blastocysts of both groups. In addition, Day-7 blastocysts of both Mito-TEMPO and CONTROL groups were individually vitrified using BO-VitriCool™-media and the Cryotop® vitrification system. Warming of these vitrified blastocysts (BO-VitriWarm™-media) was subsequently followed by post-warming culture for 72 h to determine viability rates, re-expansion rates, and hatching rates. As a result, the present study did not obtain any significant effect of Mito-TEMPO supplementation on early embryonic development or ROS levels compared to the CONTROL group. In contrast, supplementation of culture medium with Mito-TEMPO significantly (P < 0.05) enhanced re-expansion rates 32, 48, 56, and 72 h after warming compared with control embryos, respectively. Likewise, hatching rates of blastocysts previously cultured in medium supplemented with Mito-TEMPO were significantly increased at 48, 56, and 72 h after warming, respectively. Taken together, these results confirm our hypothesis that the antioxidant Mito-TEMPO reduces cryo-induced damage and exerts a positive effect on embryonic development after vitrification.
