45 SPINDLE CONFIGURATION AND DNA FRAGMENTATION OF VITRIFIED BOVINE OOCYTES AFTER IN VITRO MATURATION WITH L-CARNITINE AND/OR RESVERATROL

Abstract

After cryopreservation, oocytes may suffer morphological and functional damage, due to the high cytoplasm lipid content and to the reactive oxygen species formation. The aim of this work was to evaluate the spindle configuration and the DNA fragmentation of vitrified/warmed oocytes after in vitro maturation (IVM) in a media supplemented with l-carnitine and/or resveratrol, a lipolytic and antioxidant agent, respectively. The IVM viable COC with at least 3 cumulus cell layers and homogenous cytoplasm were randomly distributed into 4 groups: (1) control: conventional IVM media with TCM-199, epidermal growth factor, and 10% FCS; (2) L-CAR: control media supplemented with 0.6 mg mL–1 of l-carnitine; (3) RES: control media supplemented with 1 μM mL–1 of resveratrol; and 4) L+R: control media supplemented with 0.6 mg mL–1 of l-carnitine and 1 μM mL–1 of resveratrol. After 22 h of IVM, half of the COC from each group were vitrified and warmed, using the cryotop methodology. After warming, the oocytes were allowed to recover in their respective media for 2 additional hours. After 24 h of IVM, oocytes from all treatments were completely denuded and fixed and stained using specific fluorescent probes. The microtubule/chromosome configuration and the DNA fragmentation were analysed by immunocytochemistry under a fluorescent microscope (A.40FL, Carl Zeiss, Oberkochen, Germany). All statistical analyses were conducted with IBM SPSS 19 (IBM; Chicago, IL, USA). ANOVA was performed to analyse differences in meiotic spindle configuration, and the Chi-squared test was used for DNA fragmentation. The significance level was 5%. Although vitrification may cause severe oocyte damage, IVM with l-carnitine alone or in association with resveratrol was able to reduce the percentage of abnormal spindle configurations (Table 1), whereas the addition of resveratrol alone or its association with l-carnitine reduced DNA fragmentation of IVM oocytes after a vitrification/warming process. These results indicate the IVM supplementation with RES and/or L-CAR could modify oocyte composition, increasing its cryotolerance. However further studies are required to confirm the beneficial effect of these molecular interactions. Table 1.Evaluation of spindle configuration (Experiment 1) and apoptotic cell status (Experiment 2) of fresh or vitrified/warmed oocytes matured with RES and/or L-CAR