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Vertebrate reproductive science and technology

178 Evaluation of polar body extrusion following exposure of cattle oocytes to different concentrations of ethylene glycol cryoprotectant

M. L. Mphaphathi A B , M. Nkadimeng A , S. M. Sithole A C , M. D. Sebopela A C , F. V. Ramukhithi A , M. M. Tshabalala A C , A. Maqhashu D , H. O'Neill B and T. L. Nedambale A B
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- Author Affiliations

A 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;

D University of Pretoria, Department of Animal and Wildlife Sciences, Pretoria, Republic of South Africa

Reproduction, Fertility and Development 32(2) 217-217
Published: 2 December 2019


Cryoprotectant (CPA) toxicity has been recognised as a critical barrier to further advancement of cattle oocytes. Furthermore, CPA at high concentration might be toxic to oocytes, leading to osmotic shock and cell death (Arav et al. 1993). Selection of quality oocytes among a heterogeneous pool is mostly done subjectively. The use of Brilliant Cresyl Blue (BCB) stain for oocyte selection before ethylene glycol (EG) CPA toxicity test on cattle oocytes might be a useful tool. The objectives of this study were to elucidate the toxicity of EG penetrating CPA to cattle oocytes and the effectiveness of BCB on oocyte selection. Ovaries from cows of unknown reproductive status were collected from the local slaughterhouse and transported in warmed (37°C) saline water to the laboratory within 2 h of slaughter. The oocytes (n = 374) were exposed to 26 mM BCB for 90 min at 38.5°C under an atmosphere of 5% CO2. The other oocytes (n = 450) were not exposed to BCB solution or CPA (positive control: no BCB and no CPA exposure). Oocytes were classified as BCB positive (+) with a varying degree of blue cytoplasm or BCB negative (−) with no blue cytoplasm. Oocytes were exposed in EG 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% (stepwise increased CPA). The oocytes were then in vitro matured (IVM) as per treatment groups for 22 h. After maturation, oocytes were removed from the maturation medium and denuded of granulosa cells by vortexing. The polar body extrusion was evaluated with the aid of Oosight Imaging System (Hamilton Thorne) connected to an inverted research microscope. Treatment means were compared using Fisher protected t-test least significant difference. There was a drastic decline of oocytes with polar body extrusion as CPA concentration increased (P < 0.05): 40.5% (positive control, no BCB and no CPA exposure), 33.3% [(control, CPA exposure (EG 5 + 10%)], 23.1% [BCB− with CPA toxicity test (EG 5 + 10%)], 12.5% [(BCB− with CPA toxicity test (EG 10 + 20%)] and 4.9% [(BCB− with CPA toxicity test (EG 15 + 30%)]. The BCB+ groups (EG 5 + 10% and EG 10 + 20%) had significantly more oocytes with polar body extrusion (68.9% and 51.9%) compared with the positive control (40.5%), respectively (P < 0.05). In conclusion, higher EG cryoprotectant concentrations compromise oocyte polar body extrusion following IVM. We recommend that BCB be used for selection of suitable oocytes before the CPA toxicity test because of its ability to stain larger and more competent oocytes from cattle.