Effects of different fatty acids on the generation of reactive oxygen species and mitochondrial membrane potential in bovine embryos produced in vitro.

• Grant number: 24/22197-5
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: March 01, 2025
• End date: December 31, 2025
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: Claudia Maria Bertan Membrive
• Grantee: Catarina Lazarevicius Prestes
• Host Institution: Faculdade de Ciências Agrárias e Tecnológicas. Universidade Estadual Paulista (UNESP). Campus de Dracena. Dracena , SP, Brazil

Abstract

The manipulation of oocytes and embryos during in vitro embryo production (IVEP), as well as the culture conditions themselves, are well known to contribute to oxidative stress (OS). OS is characterized by an imbalance between the production of reactive oxygen species (ROS) and their removal through enzymatic or non-enzymatic systems. ROS induce cellular damage and trigger apoptotic mechanisms. The optimization of culture conditions for bovine embryos during IVEP is of significant interest. The addition of fatty acids (FAs) to the culture medium may help mitigate OS and enhance the quality of commercially produced embryos in vitro. Strategies involving polyunsaturated fatty acids (PUFAs) from the omega-6 (n-6) family, such as conjugated linoleic acid (CLA) or non-conjugated linoleic acid (LA), have been investigated by this group (FAPESP Projects 2018/24168-1 and 2022/01110-3). Oleic acid (OA), a monounsaturated fatty acid from the omega-9 family, has also been linked to the reduction of OS. The hypothesis is that supplementation with CLA, LA, or OA in the in vitro culture medium (IVC) reduces OS, contributing to improved embryonic production. Therefore, the aim is to evaluate the effects of supplementation with LA (C18:2n-6, omega-6), CLA (cis-9, trans-11 and trans-10, cis-12 isomers, omega-6), and oleic acid (OA; C18:1, omega-9) on OS, by measuring ROS levels and mitochondrial membrane potential, in embryos produced in vitro on Day 8 (D0 = day of in vitro fertilization). A 4 x 1 experimental design will be used, with four treatments (Control, 100 µM CLA, 100 µM LA, and 100 µM OA) applied at a single time point (IVC). A total of 8 repetitions will be performed. In Nelore females, ovarian follicles ranging from 2 to 10 mm in diameter will be aspirated using a 10 mL syringe attached to an 18G needle. Oocytes with homogeneous cytoplasm and multiple layers of cumulus cells will undergo in vitro maturation (IVM) in 100 µL droplets containing IVM medium with 5% fetal bovine serum (FBS), under mineral oil, in an incubator at 38.5°C in a humidified 5% CO2 atmosphere for 24 hours. Following IVM, oocytes will be fertilized in vitro (IVF) using semen from a single Nelore bull, for 18 hours under the same conditions as IVM (D0). After IVF, the presumed zygotes will be placed into 4-well plates with 700 µL of culture medium per well, containing 1.25% FBS, and supplemented with the following treatments: 100 µM conjugated linoleic acid (CLA group), 100 µM linoleic acid (LA group), or 100 µM oleic acid (OA group), in addition to the medium without added FAs (Control group). The cleavage rate will be assessed 30 hours after the start of IVF. Embryonic development will be monitored on Days 3 and 7. On Day 8, embryos will be evaluated for ROS generation using the CellROX¿ probe and mitochondrial activity using the MitoTracker¿ Red CMXRos probe. It is expected that the results of this study will contribute to the enhancement of the current culture system used in IVEP, making the commercial system more promising in terms of converting matured oocytes into embryos of sufficient quality for transfer.