83 l-Carnitine improves the bioenergetic profile of bovine embryos regardless of the protein source

Supplementation of culture media with l-carnitine (LC) is known to enhance developmental competence in terms of lipid accumulation and cryotolerance of bovine embryos, cultured either with or without estrous cow serum (ECS). The aim of the present study was to investigate the effect of LC and its interaction with two different protein sources during the in vitro culture period on the mitochondrial bioenergetic profile of bovine embryos. Therefore, a total of 1553 slaughterhouse-derived oocytes were matured for 22 h under routine procedures (TCM + 5% ECS, 5% CO₂, 5% O₂, 38.8°C, humidified air). Thereafter, cumulus–oocyte complexes were fertilized (2 × 10⁶ sperm mL⁻¹) using one bull. Presumptive zygotes were cultured in SOFaa (5% CO₂, 5% O₂, 38.8°C) under four different conditions. The first two groups were with 5% ECS, one group with 2.5 mM LC (ECS+LC) and the other one without (ECS-LC). The other two groups were supplemented with 6 mg mL⁻¹ BSA FAF (fatty acid free) either with 2.5 mM LC (FAF+LC) or without (FAF-LC). The bioenergetic profile of resulting blastocysts was analyzed by determining the oxygen consumption rate (OCR, pmol min⁻¹ per embryo) via extracellular flux analysis (mitochondrial stress test, Seahorse XFp, Agilent). The mitochondrial stress test inhibits various complexes of the respiratory chain to gain deeper insights into mitochondrial metabolism and health. OCR values were standardized relative to the values of the groups, with LC set as 100 (ratio). Statistical analyses were performed using the student t-test. No difference could be observed between the groups concerning cleavage rates (FAF-LC 84.4% vs. FAF+LC 86.3%; ECS-LC 87.9% vs. ECS+LC 90.5%) and blastocyst rates (FAF-LC 34.7% vs. FAF+LC 41.6%; ECS-LC: 44.3% vs. 48.6%). However, several parameters of the mitochondrial bioenergetic profile were significantly affected by LC supplementation, irrespective of the protein source. We observed a significant (P < 0.05) increase in mitochondrial respiration (ECS+LC 1.24 ± 0.07 vs. ECS-LC 1.0 ± 0.06; FAF+LC 1.38 ± 0.11 vs. FAF-LC 1.0 ± 0.05) and maximum respiration (ECS+LC 1.3 ± 0.07 vs. ECS-LC 1.0 ± 0.06; FAF+LC 1.45 ± 0.15 vs. FAF-LC 1.0 ± 0.02); whereas, the non-mitochondrial respiration was significantly decreased by LC independent of the protein source (ECS+LC 0.65 ± 0.07 vs. ECS-LC 1.0 ± 0.06; FAF+LC 0.58 ± 0.01 vs. FAF-LC 1.0 ± 0.23). Moreover, the ATP-linked respiration could be significantly improved by LC supplementation in the ECS groups (ECS+LC 1.21 ± 0.06 vs. ECS-LC 1.0 ± 0.08), and a strong trend (P = 0.058) toward higher ATP-linked respiration was observed for FAF groups (FAF+LC 1.27 ± 0.08 vs. FAF-LC 1.0 ± 0.08). Our results reveal that LC supplementation alleviates possible negative effects of serum during the culture period of bovine embryos and improves mitochondrial function in terms of the efficiency of ATP production. Similarly, mitochondrial function could be optimized under FAF culture conditions by LC supplementation. This means that the positive effect of LC on preimplantation bovine embryos is not limited by the presence or absence of fatty acids.