190 Evaluation of the toxicity of different concentrations of dimethyl sulfoxide and ethylene glycol cryoprotectants on immature cattle oocytes selected by Brilliant cresyl blue stain and subsequent to polar body extrusion

The objective of the study was to elucidate the toxicity of penetrating cryoprotectants (CPAs including dimethyl sulfoxide [DMSO] and ethylene glycol [EG]) on polar body extrusion in cattle oocytes. Heterogeneous cattle ovaries of unknown reproductive status (different stages of estrous cycle) were collected from a local abattoir and transported (37°C) to the laboratory within 2 h of slaughter. The aspiration method was used to retrieve oocytes from the ovaries. Oocytes (n = 821) were exposed to 26 mM brilliant cresyl blue (BCB) in basic tissue culture medium 199 (TCM-199) for 90 min at 38.5°C inside the incubator. The oocytes were classified as BCB positive (BCB+) with a varying degree of blue cytoplasm or BCB negative (BCB−) with no blue cytoplasm and subjected to toxicity testing per treatment groups. The positive control (PC) oocytes (n = 118) group were not exposed to BCB solution and CPA. The negative control (NC) oocytes (n = 486) group were exposed to CPA but not BCB solution. The CPA concentrations for DMSO + EG for toxicity test 1 (TT1) were 0%, 5%, 10%, 15%, and 20% for 3 min, followed by exposure to toxicity test 2 (TT2, for 30 s): 10%, 20%, 30%, and 40% (stepwise increases of CPAs) in TCM-199. Oocytes were then washed three times in TCM-199 + 10% fetal bovine serum (FBS). The oocytes were matured in TCM-199 supplemented with 10% FBS, LH (0.5 µg mL⁻¹), FSH (0.01 µg mL⁻¹), and estradiol (0.2 μg mL⁻¹). After IVM, oocytes were removed from the maturation medium following 22 h of IVM and denuded of granulosa cells by vortexing. The inverted research microscope (Olympus, IX71, ×10 objective) was used during evaluation of a first polar body (MII stage) extrusion in oocytes. Treatment means were compared using the Fisher’s protected t-test least significant difference. There was a decline in polar body extrusion recorded for oocytes, as the DMSO + EG concentrations were increased (P < 0.05): 51.1% (PC = no BCB and CPA exposure), 48.7% (NC = CPA exposure and no BCB), 8.2% (BCB− with CPA exposure [DMSO 5% + EG 5%]), 19.4% (BCB− with CPA exposure [DMSO 10% + EG 10%]), and 7.8% (BCB− with CPA exposure [DMSO 20% + EG 20%]). The BCB+ groups (DMSO 5% + EG 5% and DMSO 10% + EG 10%) recorded the highest number of oocytes with polar body extrusion (55.1% and 55.8%) as compared with PC (51.1%), respectively (P > 0.05). The treatment groups of oocytes exposed to DMSO 20 + EG 20% toxicity test had the lowest polar body extrusion (7.8%–20.9%) compared with those exposed to lower CPA concentrations (P < 0.05). In conclusion, higher DMSO + EG cryoprotectant concentrations compromise the cattle oocyte polar body extrusion following IVM period. It was recommended that BCB be used for selection of suitable oocytes prior to CPA toxicity tests due to its ability to stain larger and more competent oocytes from cattle.