Utilization of Rosmarinic and Ascorbic Acids for Maturation Culture Media in Order to Increase Sow Oocyte Quality Prior to IVF.

Oana-Maria Boldura,Ioan Hutu,Camelia Tulcan,Gabriel Otava,Simona Marc,Calin Mircu,Cornel Balta

doi:10.3390/molecules26237215

Abstract

The beneficial effect of antioxidant supplementation in maturation culture media of sow oocytes was evaluated by the expression quantification of apoptotic genes and the genes that ensure stability of germ cells during fertilization. The oocytes were cultivated for 44 h in conventional medium (C) or in medium supplemented with 105 µM rosmarinic acid (R) and 0.5 mM ascorbic acid (A) and classified into three quality classes by morphological observation from which the total RNA was isolated. The gene expression of Ptx3 and the apoptotic regulator p53, Bax and BCL-2 were evaluated by quantitative PCR technique. The decreased expression of the Bax gene in the A and R groups, compared to the control, indicates a protective role of antioxidants in the cells. Cell homeostasis was maintained, as reflected in the ratio of Bax/Bcl-2 in class I COCs (cumulus-oocyte complex) regardless of the experimental group, indicating minimum cellular stress. The expression of p53 genes was higher in all class III COC, but in A1 and R1 the expression was lower than in C1, and a similar Ptx-3 gene decreased significantly in groups A1, A2, A3 and R1 compared with control groups. Antioxidant supplementation showed beneficial effects on all morphological classes of pig COCs.

Highlights

Throughout recent years, swine-assisted reproductive techniques with in vitro fertilization have been used in research primarily for studies on mammalian embryogenesis, xenotransplantation, transgenesis, and genome editing
After in vitro maturation of porcine oocytes in the medium supplemented with ascorbic acid, 96.29% of class 1 COCs (A1) were matured after 44 h, 91.17% of class 2 (A2) and 12.98% of class 3 (A3), in the R group 96.42% of class 1 COCs (R1) were matured after 44 h, 63.33% of class 2 (R2) and 31.57% of class 3 (R3) compared with groups without antioxidant where we observed that 88.46% of class 1 COCs (C1), 20.58% of class COC 2 (C2) and 6.94% of class 3 COCs (C3) were matured
By comparing the effects of the two antioxidants used according to the morphological class of COCs, we noticed that the use of ascorbic acid resulted in a better maturation of COC from class 2 by 27.84% than group R, and in class 3 rosmarinic acid had a better influence with 18.59% than group A

Summary

Introduction

Throughout recent years, swine-assisted reproductive techniques with in vitro fertilization have been used in research primarily for studies on mammalian embryogenesis, xenotransplantation, transgenesis, and genome editing. Embryo technologies involve the use of assisted reproduction, of cellular and molecular biology, and genomics. Their use in research aims to solve questions about endocrine control, molecular changes and metabolic pathways that regulate embryonic development, and in animal husbandry aims to increase the number of products with a superior genotype. In vitro fertilization (IVF) has developed from an experimental science into a technology that is extensively used in bovine species [1,2], but in swine the large scale implementation of IVF techniques is still poor due to polyspermy and low embryo development, even if there are studies with promising results [3,4]

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