Estratégias para redução do conteúdo lipídico e estresse oxidativo visando otimizar o desenvolvimento pré-implantacional e a criotolerância de embriões bovinos produzidos in vitro

Abstract

The expansion of the Brazilian cattle embryo industry, driven by the widespread adoption of in vitro embryo production (IVEP) biotechnology, has positioned Brazil prominently in this field. However, despite the advances achieved in IVEP, one of the major challenges of this biotechnology remains the low resistance of embryos to the cryopreservation process. Among the factors responsible for the limited success of this technique in various mammalian species, embryonic lipid accumulation stands out. In addition to lipid accumulation, another factor impacting the quality and survival of in vitro-produced embryos is the excessive production of reactive oxygen species (ROS). ROS, naturally produced by embryos during the pre-implantation stage, can interact with the excess embryonic lipids, leading to lipid peroxidation, a phenomenon associated with functional and structural damage, compromising the integrity and permeability of the embryonic cell membrane, with direct impacts on cryopreservation survival outcomes. Thus, considering the relevance of optimizing in vitro production and cryopreservation techniques of bovine embryos in reproductive programs of dairy breeds, especially those adapted to tropical conditions, the objective of this study was to propose some supplementation strategies in IVM, focusing on reducing lipid content and oxidative stress to obtain higher quality embryos and improved rates of preimplantation development and cryosurvival. In Experiment 1, the effects of two concentrations of L-carnitine (2.5 and 5 mM), forskolin (10 and 15 μM), and zinc oxide nanoparticles (NPZNOs) (1.0 and 1.5 μg/mL) on the development, lipid accumulation, and total cell number of in vitro-produced bovine blastocysts were evaluated. In Experiment 2, one concentration of each substance (based on the results of Experiment 1) was selected to assess their effects on cryosurvival, mitochondrial activity, and ROS production of bovine embryos. The results of Experiment 1 showed that there was no significant difference (p>0.05) in embryo cleavage rates between the forskolin groups and the control, but the control showed a difference (p<0.05) in the development rate on Day 7 (59.2%) compared to the forskolin 10 μM (42.1%) and 15 μM (37.9%) groups. Cleavage and embryonic development rates did not show significant differences (p>0.05) between the control and the groups treated with Lcarnitine and NPZNOs. A significant reduction (p<0.05) in lipid accumulation was observed in the forskolin 10 μM and L-carnitine 2.5 mM treatments compared to the control. Regarding the total cell number, there was a significant increase (p<0.05) in embryos treated with forskolin 15 μM and NPZNOs 1.0 μg/mL compared to the control.In Experiment 2, no significant differences (p>0.05) were observed in cryosurvival rates48 hours after rewarming and in mitochondrial activity between the groups. However, all treatments resulted in lower ROS production (p<0.05) compared to the control group. Collectively, the data from this study provided new insights into important aspects of bovine embryo metabolism, before and after vitrification. NPZNOs, still relatively unexplored in the field of reproduction, emerge as promising molecules in vitro embryonic development and improving cryopreservation rates, mainly due to their antioxidant potential. Thus, new possibilities for the use of these molecules in enhancing the development rates of pre-implantation stage embryos are opening up.