Effects of the three-dimensional system associated or not with melatonin supplementation during oocyte maturation on in vitro production of bovine embryos

• Grant number: 23/07128-4
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: September 01, 2023
• End date: August 31, 2024
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: Claudia Maria Bertan Membrive
• Grantee: Emily Silva do Nascimento
• Host Institution: Faculdade de Ciências Agrárias e Tecnológicas. Universidade Estadual Paulista (UNESP). Campus de Dracena. Dracena , SP, Brazil

Abstract

Considering that in the in vitro embryo production (IVEP) approximately 40.8% of the oocytes matured in vitro reach embryos and that of these 33% result in pregnancy after FTET, the need to improve the technique becomes evident. The present study proposes the use of a new three-dimensional (3D) culture system, specifically using the CellFate® membrane (Biocelltis, Florianópolis, Brazil) in oocytes and embryos, in addition to the insertion of melatonin in the oocyte maturation medium (MIV) as a strategy to reduce oxidative stress in oocytes. CellFate® 3D matrices are biomaterials in hydrogel form composed of natural polymeric nanofibers with a three-dimensional organization similar to the extracellular matrix, which provides a suitable microenvironment for the growth of any cell type in a reproducible manner. So far, no studies have been carried out to evaluate the use of such a membrane in IVEP. Melatonin has antioxidant and anti-apoptotic action in oocytes and embryos, where it has shown beneficial effects on oocyte maturation, blastocyst rate, number of cells per blastocyst and rate of hatched blastocysts. In recent pilot experiments carried out by the group, during IVM when the culture medium was supplemented with 10 µM of melatonin, there was an increase in the production of embryos on D7, whereas such an increase was not verified when the same supplementation was performed in the medium of embryo culture (CIV) or in MIV and CIV. The hypothesis is that the 3D CellFate® membrane used in MIV and CIV increases the number and quality of embryos produced in vitro on D7 and that the culture system with 3D CellFate® membrane during IVM associated with the base medium for MIV supplemented with 10 µM of melatonin (Sigma M5250), followed by CIV in a 3D system, increases the quantity and quality of embryos produced in vitro on D7 (D0 = day of fertilization). The present study aims to: a) compare 2D and 3D culture systems using CellFate® membrane in MIV and/or CIV medium (Experiment 1) and b) compare 2D culture systems with MIV base medium supplemented or not with 10 µM of melatonin with the 3D culture system as MIV base medium supplemented or not with 10 µM of melatonin (Experiment 2) on the quantity and quality of embryos produced in vitro on D7 and on the abundance of transcripts related to marker genes of embryonic quality (OCT4 and CDX2), antioxidant effect (MnSOD - superoxide dismutase 2; PRDX3 - peroxiredoxin 3 and GPX1 - glutathione peroxidase 1) and anti-apoptotic effect (TP53 - tumor protein p53; BCL2 - BCL2 apoptosis regulator; BAX - BCL2 associated X and CASP3 - caspase 3).