209 L-Carnitine supplementation during in vitro maturation reduces the oxidative stress of cat oocytes

L-Carnitine (LC) is a potent antioxidant and regulates ATP production from lipids through β-oxidation, being considered an important co-factor during IVM to improve oocyte quality in several species. The domestic cat is a useful model for assisted reproduction techniques aiming for the application of endangered wild cats. The aim of this study was to evaluate the effect of LC during the IVM on lipid content, nuclear maturation, mitochondrial activity, and intracellular levels of glutathione (GSH), and reactive oxygen species (ROS) of cat oocytes. The cumulus–oocyte complexes (COCs) were recovered from sliced ovaries regardless of the oestrous cycle phase obtained in elective surgeries and selected based on cytoplasm homogeneity and cumulus cell layers (a total of 11 replicates of COCs selection and IVM, n = 269). The COCs were allocated in two IVM treatments: control (TCM-199 supplemented with 0.02 IU mL⁻¹ FSH/LH, 100 µM cysteamine, 2.2 g L⁻¹ sodium bicarbonate, 3 mg mL⁻¹ BSA, 0.25 mg mL⁻¹ sodium pyruvate, 0.15 mg mL⁻¹ L-glutamine, 0.6 mg mL⁻¹ sodium lactate, and 0.055 mg mL⁻¹ gentamicin, for 28 h at 38.5°C in maximum humidity) and LC (control IVM media + 0.5 mg mL⁻¹ LC) (Moawad et al. 2014 Hum. Reprod. 29, 2256–2268). After IVM, COCs were denuded with hyaluronidase and the lipid content was assessed by Oil Red O solution (Sigma Chemical Co.). Both LC and control were compared regarding nuclear maturation (Hoechst 33342). After that, oocytes from both groups were submitted to mitochondrial activity (MitoTracker Red, Invitrogen™, M7512), ROS (H₂DCHFDA, Invitrogen™, D399), and GSH (CMF₂HC, Cell Tracker Blue, Invitrogen™, C12881) level analyses. Fisher’s exact test was used to evaluate the MII rate. Lipid content, mitochondrial activity, ROS, and GSH levels were compared by unpaired Student’s t-test at a significance level of 5%. No difference (P > 0.05) was found between control and LC regarding the lipid content (71% vs 65%) and nuclear maturation (59% vs 56%). LC (0.88 ± 0.11 arbitrary units, A.U.) presented a lower (P < 0.05) mitochondrial activity compared with control (1.00 ± 0.03 A.U.). There was no difference (P > 0.05) regarding GSH levels between groups (C: 1.00 ± 0.38 vs LC: 0.88 ± 0.25 A.U.), but LC reduced (P < 0.05) intracellular ROS levels (C: 1.00 ± 0.64 vs LC: 0.45 ± 0.13 A.U.), and the redox balance (ROS/GSH) (C: 1.00 ± 0.74 vs LC: 0.45 ± 0.14 A.U.). Although there was no effect on nuclear maturation and on lipid content, it was concluded that LC during IVM improves oxidative metabolism by reducing the oxidative stress of cat oocytes.

Research was supported by Capes (001), FAPERJ, and CNPq.