143 Effect of different concentration of dimethyl sulfoxide cryoprotectant on oocyte maturation rate following brilliant cresyl blue exposure
M. L. Mphaphathi A B , S. M. Sithole A C , M. D. Sebopela A C , H O’Neill B and T. L. Nedambale A CA Agricultural Research Council, Animal Production, Irene, Republic of South Africa
B University of the Free State, Department of Animal, Wildlife and Grassland Sciences, Bloemfontein, Republic of South Africa
C Tshwane University of Technology, Faculty of Science, Department of Animal Sciences, Pretoria, Republic of South Africa
Reproduction, Fertility and Development 34(2) 309-310 https://doi.org/10.1071/RDv34n2Ab143
Published: 7 December 2021
© 2022 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
To date, toxicity trials have recorded unpredictable outcomes, with no consensus on the least toxic, suitable concentration, and most toxic cryoprotectant (CPA). The IVM of oocytes is an important assisted reproductive technology capable of generating mature oocytes that can develop to metaphase II. However, selection of competent oocytes for use during IVM is still a major problem. The objectives of this study were to elucidate the toxicity of dimethyl sulfoxide (DMSO) penetrating CPA to oocytes and the effectiveness of brilliant cresyl blue (BCB) on immature oocyte preselection. Ovaries from cows of unknown reproductive status were collected from the local abattoir and transported in a prewarmed (37°C) saline water to the laboratory, following slaughter. The oocytes (n = 241) were exposed to 26 mM BCB medium (Dulbecco’s PBS + 26 mM) for only 90 min at 38.5°C under an atmosphere of 5% CO2. The other oocytes (n = 197) were not exposed to BCB solution and CPA (positive control-No BCB and No CPA exposure). The other oocytes (n = 188) were exposed to CPA without BCB exposure (control-CPA exposure, no BCB). Oocytes were either classified as BCB positive (+) with a varying degree of blue cytoplasm or BCB negative (−) with no blue cytoplasm. Cattle oocytes were exposed to DMSO at different CPA concentrations as follows: Toxicity test 1 (TT1) was 0, 5, 10, and 15%, followed by exposure to TT2 as follows 10, 20, and 30% (a stepwise increase of CPA). The oocytes were then subjected to IVM as per treatment groups for 22 h. Following maturation period, oocytes were then removed from the maturation medium and vortexed for removal of cumulus cells. The oocytes polar body extrusion was evaluated with the aid of OosightTM Imaging Sytem connected to an inverted research microscope. Treatment means were compared using Fisher’s protected t-test least significant difference. The oocytes with polar body extrusion was 55.1% (positive control, no BCB, and no CPA exposure), 55.0% (control, CPA exposure- no BCB), 22.7% (BCB− with CPA toxicity test (DMSO 5 + 10%), 21.8% (BCB− with CPA toxicity test (DMSO 10 + 20%)) and 7.3% (BCB− with CPA toxicity test (DMSO 20 + 40%). The BCB + groups (DMSO 5 + 10% and DMSO 10 + 20%) had more oocytes with polar body extrusion (67.0% and 66.9%) compared with the positive control (55.1%), respectively (P > 0.05). All treatment groups of oocytes exposed to the DMSO 20 + 40% toxicity test had the lowest recorded polar body extrusion (ranged from 7.7 to 13.1%) compared with lower CPA concentrations (P < 0.05). Furthermore, all BCB− treatments groups had the lowest polar body extrusions irrespective of DMSO concentration (P > 0.05). In conclusion, a decline in oocyte polar body extrusion was recorded as DMSO concentration increased. BCB can be used to identify developmentally competent oocytes.
