137 L-carnitine improves developmental potential of bovine oocytes exposed to high lipid concentrations during in vitro maturation

Maternal obesity is associated with elevated systemic and follicular concentrations of nonesterified fatty acids (NEFA). Addition of oleic (OA), palmitic (PA), and stearic acids (SA) to medium during bovine IVM has been used as a model for oocyte maturation under obesogenic conditions and related to detrimental effects on oocyte developmental potential. Systemically, obesity leads to altered mitochondrial metabolism linked to L-carnitine insufficiency, as it serves as a co-factor for fatty acid transport into mitochondria and regulation of pyruvate oxidation. We hypothesised that L-carnitine supplementation during IVM of bovine cumulus-oocyte complexes (COCs) in the presence of high NEFA concentrations would lessen the negative effects of exposure to excessive lipids on embryonic development and oxidative stress. COCs were collected from abattoir ovaries and matured in four groups: (1) SC (solvent control, IVM medium and 5 μL mL⁻¹ ethanol), (2) LC (SC with 3 mM L-carnitine (LC)), (3) HN [high NEFA at 200 μM OA, 150 μM PA and 75 μM SA diluted in ethanol for a total of 5 μL mL⁻¹ ethanol in maturation medium (HN)), and (4) HNLC (HN and LC). After 24 h, oocytes were fertilised (Day 0). Zygotes were cultured in chemically defined medium (CDM, Olson and Seidel 2000 J. Anim. Sci. 78, 152) at 38.5°C, 5% CO₂, 5% O₂, and 90% N₂. Cleavage (Day 3) and blastocyst (Day 8) rates were assessed for a total of 1840 cultured zygotes in six replicates. To detect intracellular and mitochondrial reactive oxygen species (ROS), Day 3 embryos (n = 329) with ≥4 cells from three additional replicates were incubated in G-MOPS (Vitrolife) containing 10 μM H₂DCFDA and 0.5 μM MitoSOX (Invitrogen) for 30 min at 38.5°C. Embryos were observed with fluorescence at 100×, and grayscale images were used for fluorescence quantification using ImageJ software (National Institutes of Health). Development rates were analysed using chi-squared tests, and fluorescence intensity was analysed using Kruskal–Wallis tests with post-hoc Dunn’s tests. Cleavage rates were not significantly different among groups. Compared to SC (18%), the presence of LC reduced blastocyst development (13%; P = 0.02), as did the presence of HN (9%; P < 0.0001); however, HNLC had higher blastocyst development (13%) compared with HN (P = 0.04), demonstrating a positive interaction of LC with HN. Cytoplasmic ROS were reduced in HNLC (127.6 ± 2.8) compared with other groups (SC: 147.8 ± 3.5, LC: 143.9 ± 3.4, HN: 152.8 ± 2.8, P ≤ 0.0008); mitochondrial ROS were reduced in LC (37 ± 1.9) compared with SC (56.9 ± 2.8, P < 0.0001), and in HNLC (28 ± 1.5) compared with HN (45.1 ± 2.4) and the other groups (P ≤ 0.01). In conclusion, addition of L-carnitine improved embryonic development of oocytes exposed to high NEFA concentrations during IVM and reduced cytoplasmic and mitochondrial ROS production in Day 3 embryos. More studies are required to verify the interactions of L-carnitine and high NEFA, but findings from the present study suggest that high lipid exposure may lead to L-carnitine insufficiency in bovine oocytes, which may alter metabolic function and developmental potential.