Effects of different fatty acids on oxidative stress of bovine embryos produced in vitro

• Grant number: 24/22198-1
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: July 01, 2025
• End date: June 30, 2026
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: Claudia Maria Bertan Membrive
• Grantee: Maria Eduarda Rocha Pinto
• Host Institution: Faculdade de Ciências Agrárias e Tecnológicas. Universidade Estadual Paulista (UNESP). Campus de Dracena. Dracena , SP, Brazil

Abstract

Oxidative stress (OS), resulting from an imbalance between the generation and elimination of reactive oxygen species (ROS), is a limiting factor in the success of embryonic development. Increased OS intensity compromises the quality of oocytes and embryos, reducing the ability of oocytes to generate viable embryos. The addition of fatty acids (FAs) to cell cultures is one of the strategies to mitigate OS. Embryos can incorporate and metabolize FAs added to the culture medium, even altering their own lipid composition. The hypothesis is that supplementation of the medium with specific FAs during embryo in vitro culture (IVC) reduces OS and modifies the lipid composition of embryos, favoring embryonic production. The objective is to evaluate the effects of conjugated linoleic acid (CLA; cis-9, trans-11 and trans-10, cis-12 isomers, omega-6), non-conjugated linoleic acid (LA; C18:2n-6, omega-6), and oleic acid (OA; C18:1, omega-9) during the IVC of bovine embryos; on embryonic production, OS, and lipid composition of embryos at 8 days of development. The FAs will be diluted in DMSO to ensure that the IVC medium in all groups contains 0.20% DMSO and 2.5% fetal bovine serum (FBS). The following treatments will be applied during IVC: control with 2.5% FBS without DMSO addition (control without DMSO), control with 2.5% FBS with 0.20% DMSO (control with DMSO), 100 µM CLA, 100 µM LA, and 100 µM OA. The concentration of 100 µM is based on previous results obtained by the group from pilot dose-response experiments for CLA, LA, and OA. Oocyte collection will be performed by follicular aspiration from ovaries of Nelore (Bos taurus indicus) females and Nelore-crossed cows (Bos taurus indicus × Bos taurus taurus) slaughtered at a single abattoir. Cumulus-oocyte complexes (COCs) will be selected based on quality criteria, using only grade I and II oocytes, characterized by homogeneous cytoplasm and multiple layers of cumulus cells. After selection, COCs will undergo in vitro maturation (IVM) in standard IVM medium containing 5% FBS, in an incubator with controlled temperature (38.5°C and 5% CO¿) for 24 hours. After IVM, COCs will be fertilized with cryopreserved bovine semen from a single Nelore bull, using a Percoll® gradient for sperm selection. In vitro fertilization (IVF) will be performed in droplets of specific IVF medium for 18 hours (D0 = day of in vitro fertilization). At the end of IVF, zygotes will undergo IVC in an incubator at a controlled temperature (38.5°C and 5% CO¿), where they will receive the respective treatments for 8 days (D8), with partial medium renewal ("feeding") on D5. Cleavage rate will be evaluated 30 hours after the start of IVF, and embryonic development will be monitored on D7 and D8. On D8, embryos will be stained with ThiolTracker¿ Violet to assess OS, where fluorescence intensity is directly proportional to the intracellular concentration of reduced glutathione (GSH) in embryos; in seven experimental replicates. On D8, embryos will also be stained with Sudan Black B for a comparative analysis of lipid distribution in embryos, where staining intensity will be measured for each embryo, assessing the area and intensity of staining per volume; in seven additional experimental replicates. The expected results aim to provide insights into the effectiveness of FA supplementation in the IVC medium of bovine embryos in preventing OS and modifying lipid composition. These findings may contribute to determining improved culture conditions, enhancing the quality of embryos produced commercially in vitro. (AU)