35 Impact of two-dimensional carbons on the viability of porcine cumulus–oocyte complexes during cryopreservation

New strategies are required to enhance oocyte cryopreservation outcomes, especially in species such as the pig in which advances on the field are still lacking. Two-dimensional carbons such as graphene oxide (GO) exhibit attractive qualities including low toxicity, high surface area, and good thermal conductivity that make them excellent candidates for cryopreservation applications. In this study, the role of GO during oocyte cryopreservation was evaluated. GO was synthesized according to a modified Hummers’ method, and GO with reduced oxygen contents (rGO) was derived by heating GO to 80°C and stirring for 24 h. Both materials were characterized using scanning electron microscopy, thermogravimetric analysis, UV-Vis spectrophotometry, and x-ray photoelectron spectroscopy. Porcine cumulus–oocyte complexes (COCs) were obtained from 2- to 6-mm follicles, and their viability was assessed using SYBR green-PI staining. Only COCs with >75% viability were vitrified in groups of 10–15 using the Cryotop® method (Kitazato Corp.). To assess the effect of the carbons, Cryotop surfaces were either left as is (control groups) or covered with 10 µL of 1% (wt/vol) GO or rGO before vitrification. After thawing, the COCs were kept in porcine oocyte medium for recovery at 39°C and 5% CO₂ in a wet atmosphere. Viability was assessed immediately (0 h) and after 3 h using SYBR green-PI staining, and COCs were classified according to their live cell percentage in the following groups: >75%, 74%–50%, 49%–25%, and <25%. Results were analyzed for significance by ANOVA, and when P < 0.05, a Tukey’s multiple comparisons test was applied (IBM SPSS Statistics 21 for Windows). The results indicated that GO and rGO failed to successfully protect COCs during vitrification, as evidenced by significant damage immediately after thawing compared with the controls (>75% live cells; 62.7% ± 6.3%, vs. 21.4% ± 8.9 vs. 84.9% ± 8.1 for GO, rGO, and control groups, respectively). Furthermore, GO and rGO did not mitigate further damage during the 3-h recovery period (>75% live cells; 35.1% ± 9.8, vs. 9.0% ± 3.8, vs. 74.9% ± 10.4 for GO, rGO, and control groups, respectively). This damage likely resulted from the formation of strong interactions between the surface of the materials and the cells, which physically harmed the cell membranes during the thawing process. Future research will explore the role of GO and rGO during the recovery phase and their interactions with culture media to minimize reactive oxygen species production, aiming to determine their potential for effective use in oocyte cryopreservation.