Meiotic block in equine oocytes as an alternative for viability improvement

Author(s)

Mariana Lemos Nagib Jorge

• Document type: Master's Dissertation
• Press: Botucatu. 2023-03-20.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Medicina Veterinária e Zootecnia. Botucatu
• Defense date: 2023-01-27
• Advisor: Fernanda da Cruz Landim; Elisa Sant'Anna Monteiro da Silva

Abstract

The maintenance of equine oocytes in meiotic block, either using meiosis inhibitors or keeping them at room temperature, is beneficial for the later embryonic development. With the objective of testing the viability of maintaining oocytes in commercial embryonic culture medium available in Brazil (Botuembryo), comparing to the culture medium added with a meiosis inhibitor (Butyrolactone), before in vitro maturation (IVM), this assay was conceived. One hundred and forty-nine equine oocytes were used, separated into 3 groups: Control, Botuembryo and Butyrolactone. The Control group went straight to IVM for 24 hours, the Botuembryo and Butirolactone groups went through a pre-maturation (PM) process for 20 hours with the respective products, and then went to IVM for another 24 hours. In the Control group, moment 1 (M1) corresponds to the moment of obtaining the oocytes, while in the Botuembryo and Butyrolactone groups, M1 corresponds to the end of PM. Moment 2 (M2), in the 3 groups, was after IVM. After staining, nuclear and cytoplasmic maturation was assessed by confocal microscopy. The investigation of gene expression was performed by RT-qPCR. The following genes were analyzed in the oocytes: Bone Morphogenetic Protein 15 (BMP15), Connexin 37 (Cx37), Growth and Differentiation Factor 9 (GDF9) and Matrix Metalloproteinase 2 (MMP2); in the cumulus cells (CCs), the following genes were evaluated: Ampiregulin (AREG), Connexin 43 (Cx43), Hyalurone Synthetase 2 (HAS2), FSH Receptor (FSHR) and LH Receptor (LHR). In the evaluation of nuclear maturation, similar results were found between the treated groups and in the assessment of cytoplasmic maturation, the highest percentage of immature oocytes in M1, the highest percentage of mature oocytes in M2, and the lowest percentage of degeneration were found in the Butyrolactone group. On the other hand, gene expression data showed that there was a failure to block molecular maturation in the Butyrolactone group, with an increase in the abundance of AREG and HAS2 in M1, and a decrease in M2. In addition, superiority was observed for the Botuembryo group, between treated groups, in M1, in relation to the lower abundance of AREG, LHR and HAS2 and the higher abundance of FSHR, in M2, it presented a greater abundance of GDF9, indicating better quality of both oocytes and CCs. Although the results obtained with the use of Butyrolactone or Botuembryo are similar in relation to nuclear and cytoplasmic maturation, the molecular maturation results demonstrated the superiority of Botuembryo to maintain the quality of oocytes and cumulus cells before carrying out assisted fertilization techniques in horses. (AU)

• FAPESP's process: 20/01646-5 - Meiotic block in equine oocytes as an alternative for viability improvement
• Grantee: Mariana Lemos Nagib Jorge
• Support Opportunities: Scholarships in Brazil - Master