Post-thaw viability, developmental and molecular deviations in in vitro produced bovine embryos cultured with l-carnitine at different levels of fetal calf serum

Abstract

l-carnitine is a well-known an antioxidant that enhanced lipid metabolism. Therefore, this study investigated the influence of supplementing l-carnitine (LC) to in vitro culture medium on preimplantation development, quality, cryotolerance and transcription profile of candidate genes. Following in vitro fertilization, embryos at zygote stage were cultured with medium supplemented with LC at 1.5 mM and fetal calf serum (FCS) at 0, 2.5, 5, 7.5 and 10% of the CR1-aa culture media. Intracellular quality of produced embryos was measured using different fluorescent stains that measured reactive oxygen species (ROS), lipid and mitochondria intensities. In addition, total cell number and total apoptotic cells were counted per embryo. Quantitative expression of candidate genes was conducted to find out molecular response of embryos after treatment. Moreover, vitrification was done at day 8 of preimplantation development to evaluate post-thaw embryo viability. The results indicated improved blastocyst formation rate at day 8 of preimplantation development (day zero = day of IVF) when embryos cultured with LC supplementation at low FCS at levels of 2.5% (35.3%) and 5% (34.7%) compared to control (25.9%), LC + FCS 7.5% (26.5%) and LC + FCS 10% (28.1%) groups. The total number of blastocyst cells that were cultured with LC + FCS 2.5% and LC + FCS 5% was increased and the number of dead cells (apoptotic) was decreased compared to control counterparts. Intracellular mitochondria activity was enhanced and resulted in reduction of cytoplasmic lipid in embryos treated with LC + FCS 2.5% and LC + FCS 5% compared with other experimental embryo groups. In addition, intracellular reactive oxygen species level was reduced in LC + FCS 2.5%, LC + FCS 5% and LC + FCS 7.5% compared to control and LC + FCS 10% groups. The expression profile of genes regulating embryo quality (BCL2), metabolic activity (GLUT1, CPT2 and TFAM), lipolysis (LIPE, AMPKa1 and ACCα), resistance to stress (SOD2) and ability to induce pregnancy (IFNt) was up-regulated under low FCS (2.5% and 5%) combined with LC supplementation. On the other hand, genes regulating lipogenesis were down-regulated (ACSL3 and S1PR). It can be concluded that LC is an efficient culture media supplement when added with FCS at 2.5 and 5% which improved blastocyst development rate and quality. These improvements are due to enhanced utilization of intracellular embryo lipid that subsequently increased cryotolerance through orchestrating genes involved in various activities of bovine embryos.