138 Evaluation of dimethyl sulfoxide and ethylene glycol on fertilization rate of pig oocytes

Oocytes vitrified as cumulus cell–oocyte complexes at the germinal vesicle stage display low survival rates but maintain normal fertilization. Vitrification at this stage may avoid spindle damage, which is one of the most common alterations observed in vitrified MII oocytes. In early studies of pig IVF occurred with poor male pronuclear (PN) formation and polyspermic fertilization. The present study aim to evaluate the cryoprotectant toxicity on matured oocytes after exposure to 7.5% dimethyl sulfoxide (DMSO) + 7.5% ethylene glycol (EG) followed by 15% DMSO + 15% EG on IVF. Ovaries were collected from a local abattoir and transported to the laboratory (37°C) within an hour. Oocytes were retrieved from the ovary using a slicing technique, and follicular fluid was transferred to a 50-mL tube and placed in the Petri dish for oocytes searching. In the untreated control, good quality oocytes (n = 35 × 6 replicates) were subjected to IVM in 500 µL of North Carolina State University-23 media, covered with mineral oil, then incubated at 38.5°C for 44 h with 5% CO₂ and 100% humidity. In the toxicity group, matured oocytes (n = 35 × 6 replicates) were exposed to a cryoprotectant toxicity test (TT1) with 7.5% DMSO + 7.5% EG for 3 minutes followed by 15% DMSO + 15% EG (TT2) for 30 s. The TT1 and TT2 were prepared in Dulbecco phosphate-buffered saline supplemented with 20% fetal bovine serum. Frozen semen straw was thawed at 37°C for 1 minute before IVF. Fifty microliters of semen was added to each 50-µL drop of oocytes and IVF media (modified Tris buffer medium) and covered with 3 mL of mineral oil. The IVF dish was placed in an incubator for 24 h at 5% CO₂ at 38.5°C. Analysis of variance were used to analyse data using the GLM procedure, and treatment means were compared with the least significance difference test. After 24 h of IVF, oocytes were stained with 25 mg (0.025 g) of Hoechst 33342, and pronuclear formation was evaluated with the aid of the Oosight Imaging System (Hamilton Thorne). There was a significant difference between no pronucleus (0PN) of untreated oocytes (24.2 ± 17.2) and the combination of DMSO and EG (50.7 ± 21.6), and the total fertilization rate of untreated oocytes (75.8 ± 17.2) and the combination of DMSO and EG (50.3 ± 21.4). The results indicate that exposing oocytes to cryoprotectants after maturation induces subsequent alterations in the total fertilization rate to be lower and 0PN to be high. The combination of DMSO and EG had an effect on the pronucleus formation since the cryoprotectants have toxicity when used on pig oocytes after maturation. Further research is necessary concerning postmaturation cryoprotectant toxicity tests on pig oocytes and the different concentrations of permeating cryoprotectants.